Nanoparticle-loaded cells

ABSTRACT

The invention provides nanoparticle-loaded cells and compositions useful for improved imaging and therapy, for example radio-therapy. The invention also provides methods of manufacture of nanoparticle-loaded cells, methods of administering the nanoparticle-loaded cells, and methods for treatment and/or imaging.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/391,482 entitled “Nanoparticle-loaded Cells”, filed on Oct. 8, 2010bearing Attorney Docket No. 23738US01, and U.S. Provisional ApplicationNo. 61/391,452 entitled “Enhanced MSC Preparations”, filed on Oct. 8,2010 bearing Attorney Docket No. 23734US01, the contents of which arehereby incorporated by reference in their entireties.

This application is being co-filed on Oct. 6, 2011 with, andincorporates by reference: International Patent Application entitled“Nanoparticle-loaded Cells” bearing Attorney Docket No. 23738WO01, U.S.Non-provisional Application entitled “Enhanced MSC Preparations” bearingAttorney Docket No. 23734U502, and International Patent Applicationentitled “Enhanced MSC Preparations” bearing Attorney Docket No.23734WO01.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[Not Applicable]

MICROFICHE/COPYRIGHT REFERENCE

[Not Applicable]

BACKGROUND OF THE INVENTION

The present invention relates to cells loaded with nanoparticles andmethods of using nanoparticles for, for example, therapeutic purposesand diagnostic/analysis purposes such as imaging.

Electromagnetic radiation (e.g., X-rays) with very high-energy photonparticles has traditionally been used for the therapeutic treatment ofcertain diseases such as cancer. The high-energy radiation beam can befocused to a specific location, even deep within the body, to destroythe targeted cells. However, normal cells in the beam's path, at anydepth, are also killed. Consequently, there is always a conflict betweenthe dosages that will effectively kill the disease cells whilemaintaining a sufficient amount of normal cells for patient recovery.

Radiation enhancers have been developed which enhance the radiation doseto nearby tissues. Radiation enhancers include elements with high atomicnumbers which interact directly with the radiation beam to cause moretissue damage, for example, by scattering the radiation dose to nearbysoft tissue that would otherwise be relatively transparent to theradiation beam.

Hainfeld (U.S. Pat. No. 6,645,464) describes the loading of metalparticles into cells or membrane vesicles by placing metal seedparticles into the cells or vesicles, then chemically depositingadditional metal on the metal seed particles. Hainfeld further describesthe use of metal particles to improve imaging and therapy by theirinteraction with externally applied energy. Hainfeld does not teachcells such as mesenchymal stem cells (MSCs) loaded with nanoparticles.

Bulte et al. (“Feridex-Labeled Mesenchymal Stem Cells: CellularDifferentiation and MR Assessment in a Canine Myocardial InfarctionModel”; Academic Radiology, Vol 12, Suppl 1, May 2005) describe MRItracking Feridex-labeled MSCs. Bulte et al. do not teach goldnanoparticle-loaded MSCs and do not teach radiotherapy usingnanoparticle-loaded cells (e.g., nanoparticle-loaded MSCs).

Hainfeld et al. (Phys Med. Biol., 2004, 49:309) describe thepreferential uptake of gold particles by tumor tissues, which allowsselective killing of tumors by x-ray therapy. Hainfeld et al. do notteach the in-vitro loading of nucleated cells with nanoparticles and donot teach that gold particles have utility in radiation enhancement whenloaded into cells before administration.

Bikram (US 2010/0003197) describes MSCs transfected withsuperparamagnetic iron oxide nanoparticles carrying an anti-tumor gene.The MSCs are administered to induce a pro-inflammatory response againstthe metastatic cancer cells while the superparamagnetic iron oxidenanoparticles are used as MR contrast agents. Bikram does not teachnanoparticles comprising a substantial amount of gold or nanoparticlescomprising gold cores.

Despite several advances in therapeutic technology, promising laboratorydata has not translated into clinical results. For example, patientswith cancer such as advanced stage lung cancer still exhibit a high1-year mortality rate. What is needed in the art are nanoparticlescompositions and methods with a high therapeutic potential, that is, theability to effectively treat diseases such as cancer and inflammationwhile sparing non-diseased tissue.

BRIEF SUMMARY OF THE INVENTION

The invention, in general, provides for one or more carrier cells,carrier cell compositions, and methods of using such cells and/orcompositions (e.g., mesenchymal stem cells) having nanoparticles(“nanoparticle-loaded cells”) which are able to interact withelectromagnetic radiation or magnetic fields. According to the practiceof the present invention, it is believed that (while not being bound byany particular theory, however) the interaction of nanoparticles withelectromagnetic radiation or magnetic fields enhances energy depositionto local environments. Preferably, the nanoparticles utilized in thepractice of the present invention comprise a high-Z material.Additionally or alternatively, the interaction of those nanoparticlesand nanoparticle-loaded cells with one or more electromagneticradiations or magnetic fields provides opportunities for imaging oftissues and/or detection of various diseases, diseased cells, diseasestates and the like.

The invention also provides at least one composition comprising aplurality of nanoparticle-loaded cells (e.g., nanoparticle-loaded MSCs),for example, gold nanoparticle-loaded cells. Optionally, the compositioncomprises at least about 100,000 nanoparticle-loaded cells (e.g., goldnanoparticle-loaded MSCs). Optionally, at least about 10% of the cellsin the composition are loaded with one or more nanoparticles.Optionally, at least about 10% of the nanoparticle-loaded cells, e.g.,mesenchymal stem cells (MSCs), in the composition comprise at leastabout 100 ng of nanoparticles per cell. Optionally, as a furtheralternative, at least about 20%, about 30%, about 40%, about 50%, about70%, or about 80% of the cells are viable after hypothermic storage or afreeze-thaw cycle.

The invention also provides one or more methods of loading cells withthe nanoparticles of the present invention comprising the steps ofproviding at least one mixture of the cells and the nanoparticles andincubating the mixture, whereby the MSCs become loaded with thenanoparticles. Optionally, the method comprises the further step ofusing one or more transfection agents (e.g., protamine sulfate (SigmaAldrich, Allentown, Pa.), Bioporter® QuikEase™ (Genesee Scientific, SanDiego, Calif.) Lipodin-Pro™ (Abbiotech™, San Diego, Calif.), PULSIN™(Polyplus-Transfection™, New York, N.Y.), Proteo-Juice™ (EMD chemicals,Gibbstown, N.J.), Pierce Imject® (Thermo Scientific, Rockford, Ill.),Pierce Pro-Ject™ (Thermo Scientific, Rockford, Ill.), TransPass™ P (NewEngland Bio-Labs®, Ipswich, Mass.)) and/or nanoparticle-carrierconjugates (e.g., (Arginine)9 (Arg9), Tat protein, transferrin receptor,polyethylene glycol (PEG)). Optionally, the method can also comprise thestep of poration (e.g., electroporation) or viral infection.

The invention also provides at least one method of detection of anytissue (e.g., normal tissue, diseased tissue) comprising the step ofadministering nanoparticle-loaded cells (e.g., gold particle-loadedMSCs) to a subject and imaging the subject, or a portion thereof.Optionally, the subject or portion thereof comprises a diseased tissue.Optionally, the method comprises the step of imaging the diseasedtissue. Optionally, the diseased tissue releases MSC chemo-attractants.Optionally, the nanoparticle-loaded cells preferentially accumulate ator “home” to the diseased tissue, for example, a diseased tissue whichreleases chemo-attractants such as MSC chemo-attractants (e.g., a canceror inflammation).

The invention also provides at least one method of treating a subjecthaving one or more diseased tissues or diseased tissue types/cellscomprising the step of administering nanoparticle-loaded cells (e.g.,gold particle-loaded MSCs) to a subject, and irradiating the diseasedtissue with electromagnetic (e.g., X-ray) radiation or applying analternating magnetic field. Optionally, the nanoparticle-loaded cellspreferentially accumulate at or “home” to the diseased tissue.Optionally, the diseased tissue releases chemo-attractants (e.g., MSCchemo-attractants). Optionally, the method further comprises a firstincubation step, wherein the first incubation step is performedsubsequent to the administration step and prior to any irradiation step,whereby a therapeutically effective amount of the administered cellsaccumulates at the diseased tissue during the first incubation step. Byway of example, a therapeutically effective amount of administered cellsis one that delivers at least about 0.1% or at least about 0.5% or atleast about 1.0% or at least about 5.0% nanoparticles per gram of tumor.

Optionally, the method of treating a subject further comprises a secondincubation step, wherein the second incubation step is performed priorto repeating the first administration and first irradiation steps,whereby a substantial portion of the accumulated cells dissipates fromthe diseased tissue during the second incubation step. Optionally, themethod further comprises further repeating the administration andirradiation steps, sequentially, serially or otherwise.

In at least one embodiment, the method of treating a subject furthercomprises the step of detecting the location of the administerednanoparticle-loaded cells during the first incubation step, optionally,wherein detecting the location of the administered cells during thefirst incubation step comprises detecting said therapeutically effectiveamount of the administered cells accumulated at the diseased tissue.Additionally or alternatively, the method may further comprise the stepof detecting the location of the administered cells during the secondincubation step. Optionally, detecting the location of the administeredcells during the second incubation step comprises detecting saiddissipation of the substantial portion of the accumulated cells from thediseased tissue.

Nanoparticles of the present invention are high-Z material nanoparticlessuch as a lipid-based nanoparticle (liposome), silica nanoparticles,carbon nanoparticles, nanoparticles containing a high-Z element (e.g.gold), or combinations thereof.

Optionally, the high-Z material comprises radioenhancers and/or contrastenhancers having a metal with an atomic number of at least about 27 in amajority amount of the total radioenhancer and/or contrast enhancingcontent. Optionally, the heavy metal with an atomic number of at leastabout 27 is gold.

In one embodiment, the nanoparticles are gold nanoparticles. Optionally,the nanoparticles comprise a gold shell. Optionally, the nanoparticlescomprise a gold core. Optionally, the nanoparticles comprise a majorityof gold, or at least any of about 1%, about 5%, about 10%, or about 25%gold. Optionally, the nanoparticles comprises a diameter of less thanabout 10 nm, for example, between about 1 nm to about 5 nm, betweenabout 1.4 nm to about 2.5 nm, or are preferably about 1.9 nm (e.g.,Aurovist™ particles, Nanoprobes, Yaphank, N.Y.).

In at least one embodiment, the nanoparticles comprise a semiconductorsuch as a quantum dot. Optionally, the quantum dot comprises cadmium.

In at least one embodiment, the nanoparticles are magnetic,paramagnetic, or superparamagnetic particles. Optionally, thenanoparticles comprise a metal oxide. Optionally, the metal oxide is aniron oxide. Optionally, the superparamagnetic particles comprise aferumoxide (e.g., ferumoxides (Feridex)).

In at least one embodiment, the cells are tumor-homing orinflammation-homing cells, for example, cells which preferentiallyaccumulate at diseased tissue which releases cell chemo-attractants.Optionally, the cells are mesenchymal stem cells (MSCs), fibroblasts, orother stem cells.

In at least one embodiment, the cells are MSCs (e.g., hMSCs being “humanmesenchymal stem cells). Optionally, the MSCs are bone marrow derivedMSCs.

In at least one embodiment MSCs are isogenic MSCs. In at least oneembodiment MSCs are allogeneic MSCs. Optionally, the MSCs areadministered autologously.

In at least one embodiment, the carrier cells are additionally oralternatively loaded with an active agent (e.g., cancer therapeutic).Optionally, the active agent is a protein such as a cytokine.

Optionally, the nanoparticle-loaded cells are labeled with a targetingmoiety. Optionally, the targeting moiety targets tumor cells.Optionally, the targeting moiety is an antibody.

Nanoparticle-loaded cells can accumulate in or identify any diseasedtissue. In one embodiment, the diseased tissue is a cancer. Optionally,the cancer is lung cancer. Optionally, the cancer (e.g., lung cancer) isadvanced stage lung cancer. Optionally, the cancer (e.g., lung cancer)is a small cell carcinoma. Optionally, the cancer (e.g., lung cancer) isa non-small cell cancer. Optionally, the cancer is breast cancer orprostate cancer.

In at least one embodiment, the diseased tissue is a tissue withinflammation.

Nanoparticle-loaded cells can be administered to any subject. In oneembodiment, the subject has a tumor or inflammatory disease.

In at least one embodiment, the step of administering thenanoparticle-loaded cells comprises systemic administration, forexample, infusion (e.g., intravenous (IV) infusion).

In other embodiments, the step of administering the nanoparticle-loadedcells comprises additional routes, including, for example, subcutaneousadministration, intramuscular administration, or intraperitonealadministration.

In at least one embodiment, the step of administering thenanoparticle-loaded cells comprises direct injection, for example,injection into tissues, diseased tissues, cancer tissues, solid tumors,or the heart.

In other embodiments, the step of administering the nanoparticle-loadedcells comprises additional routes, including, for example, subcutaneousadministration, intramuscular administration, or intraperitonealadministration.

In at least one embodiment, nanoparticle-loaded cells are administeredto a subject and detected using an imaging step. Optionally, the imagingstep comprises irradiating the diseased tissue with non-therapeuticelectromagnetic radiation. Optionally, the imaging step comprisesmagnetic resonance imaging (MRI).

In at least one embodiment, nanoparticle-loaded cells are administeredto a subject having diseased tissue and the diseased tissue isirradiated with electromagnetic radiation. Optionally, theelectromagnetic radiation is x-ray radiation, for example, kilovoltageor megavoltage radiation.

In at least one embodiment, the step of irradiating comprises whole-bodyirradiation, irradiation of a diseased organ, or irradiation of a tumorsite or inflammation site.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 depicts a method of imaging in an animal model where the animalis injected with gold nanoparticles.

FIG. 2 depicts a method of imaging in an animal model where the animalis injected with nanoparticle-loaded cells.

FIG. 3 depicts a method of radiation treatment in an animal model whenradiotherapy is enhanced with nanoparticle-loaded cells.

FIG. 4 depicts fluorescence activated cell sorting (FACS) analysis ofMSCs transfected with FluoroNanogold™ (Nanoprobes, Yaphank, N.Y.) usingBioPORTER® QuikEase™ Reagent (Genesee Scientific, San Diego, Calif.).

FIG. 5 depicts FACS analysis of hMSCs transfected with FluoroNanogold™using Lipodin-Pro™ Transfection Reagents (Abbiotech, San Diego, Calif.).

FIG. 6 depicts FACS analysis of hMSCs transfected with FluoroNanogold™using PULSin™ Delivery Reagent (Genesee Scientific, San Diego, Calif.).

FIG. 7 depicts: a) Fluorescece microscopy of hMSCs transfected with 1:1FluoroNanogold™ using about 50 ug/ml protamine sulfate; b) FACS analysisof hMSCs transfected with FluoroNanogold™ using protamine sulfate; andc) FACS analysis of unmodified hMSCs (negative control).

FIG. 8 depicts FACS analysis of hMSCs transfected with FluoroNanogold™using BioPORTER® QuikEase™ Reagent (Genesee Scientific) and protaminesulfate.

FIG. 9 depicts FACS analysis of hMSCs transfected with FluoroNanogold™using Lipodin-Pro™ Transfection Reagents (Abbiotech) and protaminesulfate.

FIG. 10 depicts FACS analysis of hMSCs transfected with FluoroNanogold™using PULSin™ Delivery Reagent (Genesee Scientific) and protaminesulfate.

FIG. 11 depicts results of a chemotaxis assay (10× magnification)performed using a) unmodified hMSCs (positive control); and b) goldnanoparticle-loaded hMSCs.

FIG. 12 depicts results of a chemotaxis assay (20× magnification)performed using gold nanoparticle-loaded hMSCs.

FIG. 13 depicts population density of replated nanoparticle-loadedcells. Upper left: Unmodified hMSCs. Upper right: hMSCs loaded with 50μg/cm2 Aurovist™, about 50 μg/ml protamine sulfate. Lower left: hMSCsmodified with 50 μg/cm2 Aurovist™, 20 μg/ml protamine sulfate. Lowerright: hMSCs modified with 100 μg/cm2 Aurovist™, 20 μg/ml protaminesulfate.

FIG. 14 depicts a method of generation, isolation and cleaning ofnanoparticle-loaded cells.

FIG. 15 shows results of hypothermic storage of gold-nanoparticle loadedMSCs.

FIG. 16 depicts fluorescence microscopy and FACS analysis of hMSCstransfected with BSA Alexa Fluor® 488 (Invitrogen™, Carlsbad, Calif.)conjugate.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following definitions and abbreviations apply:

“Carrier cells” (or “cells”) means any cells which can be loaded withnanoparticles of the instant invention. Examples of useful cellsaccording to the present invention are mesenchymal stem cells (MSCs),fibroblasts, and other stem cells, e.g., hematopoietic stem cells(HSCs), or embryonic stem cells.

“Core”, as is pertains to nanoparticles, refers to the area at thecenter of the particle that is covered by at least one surface material.The core can make up any size portion of the entire nanoparticle as longas it is covered by at least one surface material. For example, the coremay comprise the center ⅕ (by diameter) of the particle while theremaining portion of the nanoparticle is made up of at least one surfacematerial covering the core. Single and multi-compartment nanoparticleshave a core as used herein.

“Exemplary” (or “e.g.,” or “by example”) means a non-limiting example.

“Freeze-thaw cycle” or “cryoprotective freeze-thaw cycle” meanscryogenic freezing followed by thawing and in vitro culturing underconditions to preserve viability, especially as taught herein accordingto the practice of the present invention.

“Heavy metals” or “high-Z elements” as used herein refer to metalelements with an atomic number of at least about 22, including, forexample, gold (Z=79), silver (Z=47), platinum (Z=78), palladium (Z=46),cobalt (Z=27), iron (Z=26), copper (Z=29), tin (Z=50), tantalum (Z=73),vanadium (Z=23), molybdenum, tungsten (Z=74), osmium (Z=76), iridium(Z=77), rhenium (Z=75), hafnium (Z=72), thallium (Z=81), lead (Z=82),bismuth (Z=83), gadolinium (Z=64), dysprosium (Z=66), holmium (Z=67),and uranium (Z=92).

“hMSCs” means human MSCs.

“HSCs” means hematopoietic stem cells.

“Imaging effective amount” means the amount of radiation (or the amountof nanoparticles) required for imaging a subject administered withnanoparticle-loaded carrier cells of the present invention. An imagingeffective amount can be determined by the skilled artisan based upon theradiation type and estimated dose enhancement based on the empiricalabsorption coefficients at different energies measured for tissue, goldand other metals. Specifically, attenuation in a material is given by:I/I o=exp(−μρx) where I is the transmitted intensity, Io is the initialintensity, μ is the mass attenuation coefficient, p is the density ofthe material and x is the thickness. Generally, but not always, animaging effective amount is less than a “therapeutically effectiveamount” of radiation.

“Majority” means any amount more than half. In the absence of a unitdescription (expressly or impliedly), the unit is to be considered byweight (i.e. weight/weight).

“Nanoparticle-loaded cell” means a cell which is physically associatedor complexed with nanoparticles. For example, a nanoparticle-loaded cellmay be a cell which comprises one or more nanoparticles localizedintracellularly (e.g., cytoplasmic or associated with a subcellularorganelle or subcellular membrane). Other examples ofnanoparticle-loaded cells include one or more cells which comprisetransmembrane nanoparticles or nanoparticles otherwise associated withthe plasma membrane. Such cell membrane association may be throughelectrostatic interaction with a membrane lipid, membrane protein, or amacromolecule (e.g., an antibody) conjugated to the plasma membrane.

“Selectively sparing” or (“selectively spared”) means the therapeuticbenefit that results from a preferential destruction of “unhealthycells” (e.g., cells, the destruction/death of which can have atherapeutic effect). Selective sparing can be demonstrated by adestruction of a higher percentage of unhealthy cells than thepercentage of healthy cells destroyed in the same treated portion.Selective sparing is compared with a similar treatment withoutadministrating the nanoparticle-loaded carrier cells.

“Therapeutically effective amount” means the amount of radiation (or theamount of nanoparticles) required for a therapeutic treatment of asubject administered with nanoparticle-loaded carrier cells of thepresent invention. Therapeutically effective amount can be determined bythe skilled artisan based upon well-understood parameters including theradiation type and estimated dose enhancement based on the empiricalabsorption coefficients at different energies measured for tissue, goldand other metals.

Nanoparticles

Nanoparticles useful according to the practice of the present inventioncomprise a material which acts as a radioenhancer and/or contrast agent.A number of elements, alloys, and compounds are known to be usefulradioenhancers, thermotherapeutic agents, and/or contrast agents. Eachof the embodiments contemplated herein can optionally comprise ananoparticle comprising gold or a nanoparticle with a gold core (unlessotherwise expressly excluded).

In at least one embodiment, the nanoparticles are radioenhancingnanoparticles, i.e., contain a radioenhancer.

In at least one embodiment, the nanoparticles are contrast-enhancingnanoparticles, i.e., contain at least one contrast agent.

In at least one embodiment, the nanoparticles are dualradioenhancer-contrast agents.

In at least one embodiment, the nanoparticles of the present inventionare high-Z material nanoparticles selected from lipid-based nanoparticle(liposome), silica nanoparticles, carbon nanoparticles, nanoparticlescontaining a high-Z element (e.g. gold), or combinations thereof.

In at least one embodiment, the nanoparticles comprise a semiconductor,e.g., a quantum dot. Optionally, the quantum dot comprises cadmium.

In at least one embodiment, the nanoparticles comprise a magneticmaterial.

In at least one embodiment, the nanoparticles comprise a diameter ofbetween about 0.8 nm and about 400 nm. Optionally, the diameter is lessthan about 10 nm, for example, between about 1 nm to about 5 nm, betweenabout 1.4 nm to about 2.5 nm, or preferably about 1.9 nm (e.g.,Aurovist™ particles). Optionally, the diameter is about 0.8 nm to about20 nm in diameter; or about 0.8 nm to about 3 nm in diameter.Optionally, the diameter is the diameter of a high-Z core portion of thenanoparticle. Optionally, the diameter is the diameter of the high-Zcontaining portion of the nanoparticle.

Nanoparticles may be provided with any organization or architecture. Inone embodiment, the nanoparticles comprise nanostars, nanoshells, ornanorods. The nanoparticles can optionally form linear, branched,cyclic, or combinations thereof of self-assembled nanostructures.

Exemplary useful high-Z materials are high-Z elements in an amountsufficient to provide contrast- and/or radio-enhancement. A high-Zmaterial can be a heavy metal in elemental form or complexes of heavymetals such as metal oxides and polyanions. Optionally, the high-Zelement has an atomic number of at least about 27. Optionally, thehigh-Z material contains an element with an atomic number of at leastabout 27 and is present in a majority amount of the total amount ofhigh-Z material present in the nanoparticle. Optionally, the high-Zmaterial is a heavy metal (e.g., gold). Other examples of high-Zmaterials are well known in the art.

Useful magnetic materials according to the present invention includeferromagnetic, ferrimagnetic, paramagnetic, and superparamagneticmaterials. Optionally, the magnetic particles comprise a metal oxide.Optionally, the metal oxide is an iron oxide. Optionally, theparamagnetic or superparamagnetic particles comprise a ferumoxide (e.g.,Feridex). Useful paramagnetic and superparamagnetic metal oxidenanoparticles are described, for example, in US2010/0003197.

In one embodiment, the nanoparticles comprise gold. Optionally, thenanoparticle comprise a gold core. Optionally, the nanoparticlescomprise gold as the primary metal, i.e. gold is the most abundant metalby weight. Optionally, the nanoparticles comprise gold in a majorityamount of the total radioenhancer and/or contrast enhancing content.Optionally, the nanoparticles comprise gold in a substantial amount ofthe total radioenhancer and/or contrast enhancing content. Optionally,the nanoparticles comprise gold in an amount greater than an inert shelllayer-amount, for example in an amount greater than nanoparticlesdescribed in US 2010/0003197. According to the present invention, goldnanoparticles loaded in carrier cells have a number of features that aredesirable for in vivo therapeutic use. For example, gold nanoparticlesmay have a high solubility, accumulate specifically in the tumor andreside in tumors longer than in the blood or muscle. In addition, goldnanoparticles may be substantially non-toxic, may have very low liveraccumulation and may be eliminated from the body predominantly throughthe kidney. Gold has the ability to form a range of sizes in thenanometer range, and is relatively inert and substantially non-toxic.

Gold nanoparticles offer several advantages in the present invention.Carrier cells such as MSCs which are loaded with gold nanoparticlestaught herein have one or more of the following unexpected properties:

-   -   viability;    -   capacity to migrate    -   capacity to preferentially accumulate or “home” to tumors and/or        inflammation;    -   capacity to proliferate;    -   capacity to differentiate;    -   any of the above properties in-vivo;    -   any of the above properties after a freeze-thaw cycle;    -   any of the above properties after hypothermic storage.

Other surprising properties of gold nanoparticles (e.g., when loaded incarrier cells) include a capability of cells to maintain the loading ofa majority (e.g., more than half) of the nanoparticles in cells for atleast about 1 day, at least about 2 days, or at least about 5 days.

Another property of gold nanoparticles (e.g., when loaded in carriercells) according to the present invention include the low level ofimmunogenicity, the lack of an increase in immunogenicity, or totalnon-immunogenic nature.

In at least one embodiment, the nanoparticles comprise a high-z elementin the core. Examples of such nanoparticles are solid (singlecompartment) nanoparticles and nanoparticles with a core and a shellcontaining another material. Optionally, the metal core is a solid metal(e.g. gold) core. Optionally, the nanoparticles comprise a heavy metalcore and a surface or shell layer of another material. Optionally, themetal core consists primarily of one metal such as gold, silver, iron,platinum, palladium, iridium, tungsten and others listed above. Inanother embodiment, the metal core is a mixture or an ordered,concentric layering of such metals, or a combination of mixtures andlayers of such metals. For example, alloys can be formed duringsynthesis by having two or more metal sources available for reduction.Alternatively, the metal core can be composed of two or more concentricshells of different metals. These are produced by forming the centralmetal particle, then depositing on it an additional layer of a differentmetal by electroless plating. Electroless plating, or autometallography,or metal enhancement, is performed by combining the starting metalparticles with a source of ions of the same or a different metal and areducing agent. The starting metal particles act catalytically toaccelerate metal deposition from the solution, as opposed to extraneousmetal deposition caused by autonucleation. By supplying only limitedamounts of reducing agent or metal ions, the thickness of the metalcoating can be controlled. Varying the time of the reaction is anotherway to control the deposited amount.

In one embodiment, the nanoparticles comprise a metal, metal alloy, orlayers of metals. Alloyed or layered metal particles optionally have anumber of advantages over nanoparticles of one metal. For example,alloyed or layered metal particles may have better pharmacokineticcharacteristics. The toxicity of a more toxic metal can be controlled bycoating or alloying with another metal that is non-toxic. For example,lead nanoparticles can be coated with a chemically inert and non-toxiclayer of gold. In additional, because various metals interact withradiation differently, a wider range of choices for enhancement of doseis available with alloyed or layered metal particles. Moreover, othermetals may be less expensive than gold, making some choices morecommercially attractive.

In accordance with the present invention, non-metal elements can also bepresent in a metal core, such as silicon, oxygen, and phosphorus. Anexample is a metal heteropolytungstate. By way of further example, ametal heteropolytungstate can have the formula, W₁₂O₄2Si.

The metal may be surrounded by a surface or shell layer of anothermaterial that is either covalently bound to the core or held to the coreby non-covalent forces such as charge, hydrophobic forces, van der Waalsinteractions, or a combination thereof.

Surface layer materials suitable for use in accordance with the practiceof the present invention include molecules containing, for example,sulfur, phosphorus or amines (e.g., phosphines, phenanthrolines, silanesand organo-thiols) since sulfur, phosphorus and amines can form bondswith surface metal atoms. The thiol group can be linked to a sugarcompound, such as glucose, a sugar oligomer or polymer.

Other surface layer materials suitable for use in accordance with thepresent invention include synthetic polymers, proteins, antibodies,antibody fragments, peptides, nucleic acids, carbohydrates, lipids,drugs, and other compounds, which can bind to the metal core bynon-covalent interactions such as charge, hydrophobic or van der Waalsinteractions, or bind to the metal core by covalent interactions.

The surface layer material may be present during the reduction processor pre-attached to metal atoms, either becoming incorporated into theshell in situ or being added after the metal particle is formed.Alternatively, a metal nanoparticle with a first surface layer materialis formed, which then exchanges some or all of the surface material witha second surface material. This exchange process may in some cases behastened by heating in the presence of excess second shell material.Metal particles with the original shell material or the second shellmaterial can be linked via chemical reactions to virtually any othermolecule desired, be it a lipid, antibody, carbohydrate, nucleic acid,peptide polypeptide, drug or synthetic molecule.

A shell layer may be provided, for example, to contribute to theparticle's properties, such as solubility, toxicity, affinity, andpharmacokinetics (biodistribution in animals as a function of time). Forexample, it is known that gadolinium ions are highly toxic, but whencomplexed with an organic shell of DTPA(diethylenetriaminepentaacetate), they are non-toxic, and commonly usedas a MRI contrast agent.

In at least one embodiment, gold nanoparticles are provided which areabout 1 nm to about 3 nm in diameter (e.g., 1.9 nm) and optionallycomprise thioglucose molecules as an organic shell material. The goldnanoparticles are useful, for example, as radioenhancers and/or contrastagents.

In at least one embodiment, the nanoparticles comprise polyanions ofmetals complexed with quaternary ammonium salts or covalently coatedwith an appropriate surface layer material for use in radiationenhancement. Polyanions are nanoparticle structures or metal-oxygenclusters formed by metals such as tungsten, vanadium, and molybdenum inan aqueous solution, which are characterized by metal-oxygen bondsrather than metal-metal bonds typical of nanoparticles of gold, silver,platinum, and palladium. Such polyanion particles are also referred toas heteropolyanions where a mixture of elements is present. An exampleof heteropolyanions is M₁₂O₄₂X^(n)—, where M=V, Mo, or W, and X=Si, P,V, Co, or B, and n>1. Other larger stable clusters are known such asones containing M₁₈ and M₃₀. Heteropolyanions may be complexed withquaternary ammonium salts to provide stable forms that are tolerated invivo, and are therefore useful and safe for use in enhancing the effectsof radiation therapy. Forming a complex with quaternary ammonium saltscan shield such high charge and thus reduce the toxicity ofheteropolyanions.

Nanoparticles comprising a metal (e.g., metal core) can be made usingtechniques known in the art, e.g., those described in U.S. Pat. No.5,521,289 and U.S. Pat. No. 6,369,206, the teachings of which areincorporated herein by reference. For example, gold particles may beformed by reducing a gold ion source with a reducing agent such asphosphorus, borane, citrate, sodium borohydride, ionizing radiation,alcohol, aldehyde, or other reducing agent.

The size of metal cores can be controlled by using a certain type ofreducing agent, including additional components in the reductionreaction that affect particle size, or altering the amounts andconcentration of component reagents. Alternatively, the size of metalcores can be controlled by taking a small completed nanoparticle anddepositing additional metal by autometallography.

Enhancement of Radiation

According to the present invention, nanoparticles may be provided whichcomprise a radioenhancer and interact with radiation to enhance energydeposition to local tissue.

Useful radioenhancers include elements or other materials which exhibita high degree of interaction with (e.g., absorption and/or scatteringof) therapeutic radiation (e.g., relative to soft tissue) and enhancelocal energy deposition, for example, to surrounding soft tissue.Nanoparticles interact with radiation and scatter energy, for example,by the photoelectric effect, compton scattering, and pair production,although the photoelectric effect generally dominates. The interactionof radioenhancers with radiation enhances local energy deposition by theproduction of secondary electrons, alpha particles, Auger electrons,ionizations, fluorescent photons, and free radicals, for example.Examples of radioenhancers include high-Z materials and are well knownin the art.

Useful radioenhancers include those agents known in the art as contrastagents and include elements or other materials which exhibit a highdegree of interaction with imaging radiation (e.g., radiopaquematerials) and/or are susceptible to magnetic fields used in MRI (e.g.,relative to soft tissue), wherein the interaction is detectable viaimaging. Examples of contrast agents include magnetic materials such asparamagnetic and superparamagnetic materials. Other examples are wellknown in the art.

In many cases, nanoparticles of the present invention are useful as bothradioenhancers and contrast agents due to interaction of, for example,many heavy metals with both radiation and magnetic fields. Examples ofdual radioenhancer-contrast agents are well known in the art.

Carrier Cells

According to the present invention, any cell type may be loaded withnanoparticles.

In at least one embodiment, the cells are homing cells thatpreferentially accumulate at diseased tissue, for example, inflammationand cancer tissue. Examples of homing cells include mesenchymal stemcells (MSCs), hematopoietic stem cells (HSCs), and fibroblasts.

In at least one embodiment, the cells comprise MSCs. Optionally, theMSCs are bone marrow-derived (BM) MSCs. MSCs and BM-MSCs are described,for example, in U.S. Pat. No. 6,863,900, US2007/0253931, U.S. Pat. No.6,030,836, U.S. Pat. No. 6,387,367, U.S. Pat. No. 6,875,430,US2009/0214493, U.S. Pat. No. 5,908,782, U.S. Pat. No. 7,029,666, U.S.Pat. No. 5,486,359, WO/2008/042174, and WO/2010/019886.

Optionally, the MSCs are from preparations according to U.S. ProvisionalPat App No. 61/391,452 entitled “Enhanced MSC Preparations”; filed 8Oct. 2010 and “Enhanced MSC Preparations”; filed as a U.S. patentapplication and a P.C.T. application claiming priority to 61/391,452 andbeing filed on or about 8 Oct. 2011, each of which is incorporatedherein by reference.

MSCs are especially useful cells according to the present invention.MSCs may demonstrate homing to cancerous (e.g., lung cancer) and injured(e.g., inflamed) tissue and have reduced immunogenicity. Without beingbound by theory, it is believed that MSCs migrate to tumor tissuesand/or injured tissue through chemo-attractants, i.e., factors secretedby the diseased tissue, such as growth factors, cytokines, andchemokines.

Fibroblasts can be used as carrier cells and are described, for example,in U.S. Pat. No. 7,491,388. Fibroblasts may exhibit disease-homingproperties similar to MSCs. Accordingly, fibroblasts may be provided,for example, as an alternative to MSCs in any embodiment taught herein.

HSCs can be used as carrier cells. HSCs are described, for example, inU.S. Pat. No. 6,030,836, US 2007/0134208, and US 2005/0054097. HSCs areoptionally CD34 positive. HSCs may exhibit disease-homing propertiessimilar to MSCs. Accordingly, HSCs may be provided, for example, as analternative to MSCs in any embodiment taught herein.

Surprisingly, cells such as MSCs provide superior vehicles fornanoparticles, for example, to enhance radiation therapy and/or imagingcontrast.

Carrier cells such as MSCs have one or more of the following unexpectedproperties when loaded with nanoparticles of the present invention:

-   -   viability;    -   capacity to migrate;    -   capacity to preferentially accumulate or “home” to tumors and/or        inflammation;    -   capacity to proliferate;    -   capacity to differentiate;    -   any of the above properties in-vivo;    -   any of the above properties after a freeze-thaw cycle; and    -   any of the above properties after hypothermic storage.

Other surprising properties of carrier cells (e.g., MSCs) loaded withnanoparticles according to the present invention include the capacity ofthe cells to maintain the loading of a majority (e.g., more than half)of the nanoparticles in a cell for at least about 1 day, at least about2 days, or at least about 5 days.

Another property of carrier cells (e.g., MSCs) loaded with nanoparticlesaccording to the present invention include the low level ofimmunogenicity, the lack of an increase in immunogenicity, or totalnon-immunogenic nature.

Carrier cells, when loaded with the nanoparticles of the presentinvention are optionally viable at a level at least about 20%, about30%, about 40%, about 50%, about 70%, or about 80% for at least about 24hours.

Cell Loading

According to the present invention, cells are loaded withradio-enhancing and/or contrast-enhancing nanoparticles. Cells may beloaded with nanoparticles by any method known in the art for loadingcells with agents. Loading may be accomplished, for example, byendocytosis, diffusion, active transport, injection, transfection agent,and/or bombardment.

In general, loading may be accomplished by providing a mixture of cells(e.g., MSCs) and nanoparticles (e.g., gold nanoparticles) and incubatingthe mixture, whereby cells become loaded with the nanoparticles.

Exemplary methods of nanoparticle cell loading include membranepermeabilization, transfection agent mediated loading, conjugation orcomplexation to a carrier molecule, direct injection, and bombardment.

In one embodiment, cell loading comprises the use of a transfectionagent. Optionally, the transfection agent comprises a lipid (e.g.,Lipodin-Pro™ reagent), liposome, or polymeric transfection agent.

In one embodiment, a transfection agent is an ionic (e.g., cationic)transfection agent. Optionally, an ionic transfection agent is acationic peptide (e.g., protamine sulfate), cationic lipid (e.g.,BioPORTER® Protein Delivery Reagent), or cationic amphiphile (e.g.,PULSin™ Delivery Reagent). Cationic agents such as poly-L-lysine work,for example, by coating the nanoparticles through electrostaticinteractions and bind to the cell membrane, while inducing membranebending, following which the nanoparticle is endocytosed, for example,as described in Bulte et al. (“Feridex-Labeled Mesenchymal Stem Cells:Cellular Differentiation and MR Assessment in a Canine MyocardialInfarction Model”; Academic Radiology, Vol 12, Suppl 1, May 2005). Othermethods involving the use of transfection agents are described, forexample, in US2010/0003197. Commercially available transfection agentsinclude, for example, Proteo-Juice™, Pierce Imject®, or PiercePro-Ject™, and TransPass™ P.

In one embodiment, cell loading comprises conjugating or complexing ananoparticle to a cell-penetrating carrier, for example, a cellpenetrating peptide or other molecule known to carry conjugated agentsacross membranes. Exemplary cell-penetrating carriers include Arg9, Tat,transferrin, protamine sulfate, and PEG. Useful Tat peptides and othercarriers are described, for example, in US 2002/0151004.

In one embodiment, cell loading comprises a step of cell membranepermeabilization. Optionally, cell loading comprises poration, orcausing cell membranes to temporarily become porous. Exemplary porationmethods include electroporation, sonoporation, and the like.Sonoporation is described, for example, by Miller, et al., 1998,Ultrasonics, 36: 947-952. Electroporation can be performed, for example,by mixing cell (e.g., MSCs) with nanoparticles and placing the mixturebetween electrodes such as parallel plates. Then, the electrodes areactivated to apply an electrical field to the cell/nanoparticle mixture.The electric field generated between the electrodes causes the cellmembranes to temporarily become porous, whereupon nanoparticles enterthe cells.

In at least one embodiment, cell loading comprises viral infection.Optionally the nanoparticles are bound to the protein coat of the virus.Optionally the nanoparticles are incapsidated by the viral proteinshell. With the teachings provided herein, the skilled artisan is nowable to apply relevant incapsidation technologies as described, forexample, by Loo et al. (J Am Chem. Soc. 2006 Apr. 12; 128(14):4502) andviral conjugation methods of Taeng et al. (See Nat. Nanotechnol. 2006October; 1(1):72-7).

In at least one embodiment, cell loading comprises particle bombardment.Particle bombardment entails, for example, coating gold particles withthe nanoparticles, dusting the particles onto a 22 caliber bullet, andfiring the bullet into a restraining shield made of a bulletproofmaterial and having a hole smaller than the diameter of the bullet, suchthat the gold particles continue in motion toward cells in vitro and,upon contacting these cells, perforate them and deliver the payloadnanoparticles to the cell cytoplasm. In an alternative example, thenanoparticles themselves (e.g., gold nanoparticles) are directly dustedon the bullet.

Carrier cells can also be loaded with nanoparticles as described inUS2010/0003197.

Carrier cells can be loaded on a per cell basis with at least any ofabout 0.05 atto grams (“a.g.”), about 0.5 a.g., about 5 a.g., about 50a.g., or about 500 a.g. of nanoparticles.

Carrier cells can be loaded on a per cell basis with at least any ofabout 1, about 10, about 100, about 1,000, or about 10,000 nanoparticlesper carrier cell.

A preparation of cells, according to the present invention, can comprisecarrier cells wherein at least any of about 5%, about 10%, about 25%, orabout 50% of the cells are carrier cells loaded as taught herein (i.e.,per cell basis with any of at least about 1, about 10, about 100, about1,000, or about 10,000 nanoparticles per carrier cell.).

Administration

One of ordinary skill in the art can readily ascertain a wide variety ofmethods of administration of nanoparticle-loaded cells.Nanoparticle-loaded cells may be administered, for example, systemicallyor locally.

In one embodiment, the cells are administered by intravenous (IV)injection. IV injection is well suited to delivery ofnanoparticle-loaded cells to the vascular system of animals such ashumans, primates, mammals, or other non-human animals, and is especiallyuseful where the target tissue is a tumor or an inflamed tissue.

In one embodiment, the cells are administered by intratumoral or directtissue injection. Such direct administration may be used in order toreduce the concentration of cells in other tissues and achieve a highconcentration in, for example, tumor or inflamed tissue.

Diseases

In one embodiment, nanoparticle-loaded cells are administered inconjunction with radiotherapy, thermotherapy, and/or imaging of asubject having a disease. Any disease may be treated and/or imaged usingnanoparticle-loaded cells of the invention.

Diseases that can usefully be treated and/or imaged include, forexample, cancers, inflammatory diseases, cardiac diseases, neurologicaldiseases, and other conditions with an inflammatory component.

In one embodiment, the disease is a cancer. Optionally, the cancercomprises a solid tumor. Optionally, the cancer comprises a solubletumor. Optionally, the tumor is a primary tumor. Optionally, the tumoris a secondary tumor. Optionally, the cancer is metastatic. Optionally,the cancer is advanced stage cancer. Optionally, the cancer is lungcancer. Optionally, the cancer is a small cell cancer. Optionally, thecancer is a non-small cell cancer. Other cancers that can usefully betreated and/or imaged by the present invention include hematologicalcancers.

In one embodiment, the disease that is treated and/or imaged is a lungcancer. Optionally, the lung cancer is small cell lung cancer.Optionally, the lung cancer is non-small cell lung cancer (NSCLC).Optionally, the lung cancer is advanced stage lung cancer. Optionally,the lung cancer is a primary lung cancer.

Exemplary inflammatory diseases that can usefully be treated and/orimaged by the present invention include, for example, Acne vulgaris,Asthma, Autoimmune diseases, Chronic prostatitis, Glomerulonephritis,Hypersensitivities, Inflammatory bowel diseases, Pelvic inflammatorydisease, Reperfusion injury, Rheumatoid arthritis, Sarcoidosis,Transplant rejection, Vasculitis, Interstitial cystitis. Any other acuteand chronic diseases and conditions, which characterized by the presenceof an inflammatory component, can be treated and/or imaged by thepresent invention. Such conditions include but are not limited to acutetrauma.

Exemplary Inflammatory bowel diseases that can usefully be treatedand/or imaged by the present invention include, for example, Crohn'sDisease and Inflammatory Bowel Disease.

Methods of treating cancer (e.g., lung cancer, or advanced stage lungcancer) using nanoparticle-loaded cells of the present inventionunexpectedly provide one or more of the following results, for example,compared to prior art treatments such as high-Z material-enhancedradiotherapy:

-   -   substantially reduce the mortality rate (e.g., one-year        mortality rate) of patients, for example, any of:        -   to less than about 80% for about 1 year after diagnosis, and            about 94% for about 5 year after diagnosis considering all            types of lung cancer at all stages;        -   the 1 year mortality rate to less than about 100% for stage            IV recurrent NSCLC, to less than about 65% to about 70% for            stage IIIb-IV NSCLC.        -   the 5 year mortality rate to less than about 70% to about            85% for stage IIIa, to less than about 50% to about 60% for            stage II NSCLC, and to less than about 30% to about 40% for            stage I NSCLC.    -   selectively kill diseased cells, i.e., substantially reduce the        death of healthy cells compared to diseased cells;    -   substantially reduce the number of treatment sessions and/or        treatment time required for therapeutic results;    -   substantially reduce recovery time;    -   substantially reduce the volume of tumors, for example, solid        tumors and/or soluble tumors;    -   substantially reduce the amount of radioenhancer required for        therapeutic results; and    -   substantially reduce chemical toxicity (i.e., non        radiation-based) toxicity.

When used to treat cancers or other conditions involving an inflammatoryresponse, the nanoparticle-loaded cells of the present inventionunexpectedly provide remarkable homing to the inflammation and induceonly minimal or no deleterious immunogenic response.

Therapy

Radiation Therapy

Nanoparticle-loaded cells of the present invention may be used toenhance the local dose of therapeutic radiation. The radiation sourcemay be any known in the art to be useful for treating diseased tissue.

The therapeutic radiation used in conjunction with thenanoparticle-loaded cells of the present technology may comprise, forexample, the same therapeutic radiation used in conventional therapiesthat lack the nanoparticle-loaded cells of the present technology.

The radiation may comprise, for example, x-rays, visible light, lasers,infrared, microwave, radio frequencies, ultraviolet radiation, and otherelectromagnetic radiation at various frequencies. Various other sourcesmay be employed, for example, electrons, protons, ion beams, andneutrons.

The radiation may comprise photo-thermal therapy with infrared or nearinfrared absorption by the nanoparticle-loaded cells of the presentinvention.

The use of radioenhancers in radiotherapy is known in the art. Goldnanoparticles, for example, have been shown to accumulate in the tumorarea, where they enhance local energy deposition of therapeuticradiation. However, this accumulation may only be marginal relative tothe levels of gold nanoparticles distributed through the other tissuesin the body. Surprisingly, however, nanoparticle-loaded cells of thepresent invention provide superior ablation of diseased tissue. Withoutbeing bound by theory, the present inventors believe thatnanoparticle-homing cells (e.g., MSCs) preferentially accumulate atdiseased tissue such as cancer or inflammation, thereby targetingcontrast-enhancing nanoparticles to the microenvironment of the diseasedtissue. The superior properties of the instant nanoparticle-loaded cellsare surprising, for example, because the cell membrane provides abarrier to any inherent property of nanoparticles to be taken up bydiseased tissue such as cancer. Furthermore, although cells such as MSCshave been known to accumulate under certain conditions in some diseasedtissue, it is surprising that such ability to accumulate persists or iseven enhanced in nanoparticle-loaded cells.

It is further surprising that MSCs take up nanoparticles which aresmaller than about 2 nm. Cancer cells are not known for the capacity totake up nanoparticles of a size, for example, of less than about 2 nm.Thus, the methods of the present invention provide a means to enhanceradiation therapy. In situations where the carrier cells die (e.g., withtime, post administration), the nanoparticles can be released inproximity to the cancer cells and continue to enhance radiation therapy.

In one embodiment, the radiation source comprises x-rays. Optionally,the x-rays comprise kilovoltage or megavoltage radiation. Optionally,the radiation source is a low energy x-ray, for example, of less thanabout 400 keV. Optionally, the radiation source is a high-energy x-rayof at least about 400 KeV, for example, up to about 25 MeV.

A number of interactions occur when a high Z material is subjected tox-rays. The primary beam may interact with the nucleus or electrons ofthe nanoparticle atoms or molecules (e.g., heavy metals with high Z).The interactions can be in the form of, for example, Compton scattering,elastic (e.g., Rayleigh) scattering, pair production, the photoelectriceffect, or a combination thereof.

The choice of the radiation energy can be determined taking intoconsideration various factors including, e.g., the type and location ofthe target tissue. In the presence of low energy x-rays (e.g., less than100 keV), the photoelectric effect is the predominant form ofinteraction, and the interactions with high Z material (e.g., heavymetal) nanoparticles are substantially stronger as compared to thosewith tissue (e.g., soft tissue) materials which typically have a low Znumber. With higher energy x-rays, the differential effects of theradiation (i.e., high Z nanoparticles v. tissue) may be lesssignificant; yet such higher energy sources provide energy which maypermit electrons, ejected from the K or L shell of the high Z element,to traverse adjacent cells and impart a damaging effect.

In one embodiment, x-rays of about 250 kVp (where “p” stands for thepeak, or greatest photon energy) used in conjunction with goldnanoparticles have stronger killing effects on tumor cells than aradiation source of about 100 kVp. However, for x-rays with energylevels far above the K or L shell excitation energy (e.g., >about 400keV), the cross-section for creating a photoelectron may diminish.

Optionally, such high-energy x-rays are particularly useful for treatinga target tissue deep (e.g., about 8 to about 11 cm) below the bodysurface. Traditionally, such high-energy x-rays are not a desirableoption in implementing high Z dose enhancement radiation therapy,because the absorption coefficient differences between a high Znanoparticle and tissue are believed to be much smaller than for lowenergy photons. However, a higher energy photon beam may degrade as itprogresses into tissue, and may result in a lower energy component andsecondary low energy particles generated from the tissue, includingsecondary electrons, fluorescent photons, Auger electrons, and the like.The low energy components and particles can then interact in a morefavorable way with the high Z nanoparticle, giving a greaterdifferential effect to the high Z material vs. tissue.

In still another embodiment, microbeam arrays of x-rays, now typicallyproduced at synchrotrons, are used in practicing the methods of thepresent invention. Microbeam arrays or “microbeams” are beams ofradiation that have been segmented into stacked sheets with no incidentradiation between them. This is usually accomplished by taking acollimated source and passing it through a multislit collimatorconsisting of alternating transparent and opaque lines. However, thewidth of the microbeams is typically about 20 to about 80 microns wide,and the “dead space” between them is typically about 50 to about 800microns wide. This form of radiation has been shown to spare normaltissue while being damaging to tumors. Having a nanoparticle-loaded cellproximal to the tumor would accentuate the microbeam effect.

In another embodiment, a radioactive isotope is used as the radiationsource in conjunction with nanoparticle-loaded cells in cancer treatmentor in other applications of tissue ablation. Optionally, the radioactiveisotope is an isotope of iodine (e.g., I-125, t_(1/2)=60.1 days),palladium (e.g., palladium-103, t_(1/2)=17 days), iridium (e.g.,iridium-192), or cesium (e.g., cesium-137). Optionally, the radioactiveisotope is a high dose rate isotope (e.g., iridium-192). Optionally, theradioactive isotope is a non-high dose rate isotope. Treatment times forhigh dose rate isotopes may be, for example, in the range of severalminutes, as compared to non-high dose rate isotopes which may have atreatment time of substantially longer.

A method of irradiating with a radioactive isotope may include, forexample, packaging radioactive isotopes into small metal tubes or“seeds” (typically about 5×0.5 mm) and implanting the seeds in orproximal to lung, brain, prostate or other tumors. These implantsprovide radiation locally over a period related to the isotope'shalf-life. This implant approach is also referred to as “brachytherapy”.In an alternative example, radioactive isotopes are fed throughcatheters, which are placed in and/or around a tumor.

Other Therapeutic Methods

Other methods of delivering energy to nanoparticles are alsocontemplated. For example, in any embodiment of the present invention,the step of irradiating nanoparticles with radiation can be replacedwith a step of applying an alternating magnetic field. The use of suchan alternating magnetic field is known as thermotherapy. Thermotherapyinvolves applying an alternating magnetic field to provide energy toreorient the magnetic moment of nanoparticles such as paramagneticnanoparticles. This magnetic energy, when dissipated, is converted tothermal energy, which results in destruction of nearby diseased tissue.In addition to causing changes in the magnetic moments, this energy canforce the nanoparticles to physically rotate, producing additional heat.Frictional heating, however, generally contributes much less thanmagnetic heating to the particles' total heat generation. Particles withdiameters of, for example, about 10 nm or less, typically demonstratesuperparamagnetic properties. The magnetic moments of superparamagneticnanoparticles are randomly reoriented by the thermal energy of theirenvironment and do not display magnetism in the absence of a magneticfield.

Imaging

According to the present invention, nanoparticle-loaded cells of thepresent invention are useful in methods of imaging. Useful methods ofimaging include x-ray imaging and magnetic resonance (MR) imaging.

The use of contrast agents in imaging techniques is known in the art.Surprisingly, however, nanoparticle-loaded cells of the presentinvention provide superior detection and/or imaging of diseased tissue.Without being bound by theory, the present inventors believe that thesuperior properties of the instant nanoparticle-loaded cells topreferentially accumulate at diseased tissue, such as cancer orinflammation, provide a basis for unexpected specificity incontrast-enhancement of the diseased tissue.

In one embodiment, imaging comprises magnetic resonance imaging (MRI).To obtain an image of an organ or tissue using MRI, a subject is placedin a strong external magnetic field and the effect of this field on themagnetic properties of the protons (hydrogen nuclei) contained in andsurrounding the organ or tissue is observed. The proton relaxationtimes, termed T1 and T2 are of primary importance. T1 (also called thespin-lattice or longitudinal relaxation time) and T2 (also called thespin-spin or transverse relaxation time) depend on the chemical andphysical environment of organ or tissue protons and are measured usingthe Rf pulsing technique. This information is then analyzed as afunction of distance by a computer, which uses it to generate an image.

In order to achieve effective contrast between MR images of thedifferent tissue types in a subject, it has long been known toadminister to the subject MR contrast agents (e.g., paramagnetic metalspecies) which effect relaxation times of the MR imaging nuclei in thezones in which they are administered or at which they aggregate.Contrast enhancement has also been achieved by utilizing the “Overhausereffect” in which an electron spin resonance (ESR) transition in anadministered paramagnetic species (hereinafter an OMRI contrast agent)is coupled to the nuclear spin system of the imaging nuclei. TheOverhauser effect (also known as dynamic nuclear polarization) cansignificantly increase the population difference between excited andground nuclear spin states of selected nuclei and thereby amplify the MRsignal intensity by a factor of a hundred or more allowing OMRI imagesto be generated rapidly and with relatively low primary magnetic fields.Most of the OMRI contrast agents disclosed to date are radicals whichare used to effect polarization of imaging nuclei in vivo.

In one embodiment, imaging comprises radio-imaging, for example, x-rayimaging. Briefly, transmitted radiation is used to produce a radiographbased upon overall tissue attenuation characteristics. Radiation (e.g.,x-rays) passes through various tissues and is attenuated by scattering,i.e., reflection, refraction or energy absorption. However, certain bodyorgans, vessels and anatomical sites exhibit so little absorption ofradiation that radiographs of these body portions are difficult toobtain. To overcome this problem, radiologists routinely introduce anradiation absorbing medium containing a contrast agent into such bodyorgans, vessels and anatomical sites.

In one embodiment, X-ray imaging comprises Computed Tomography (CT). CT,also known as computed axial tomography or computer-assisted tomography(CAT) and body section roentgenography, is a medical imaging methodemploying tomography where digital processing is used to generate athree-dimensional image of the internals of an object (or subject) froma large series of two-dimensional X-ray images taken around a singleaxis of rotation.

Cryopreservation and Hypothermic Storage

Nanoparticle-loaded carrier cells of the present invention may be usedfresh or may be preserved for a period of time. By way of example,preservation methods are demonstrated in Example 20 (i.e., bycryopreservation) and Example 21 (i.e., by hypothermic storage).

In one embodiment, nanoparticle-loaded carrier cells are cryopreservedby freezing (e.g., at about −80° C. or colder). Freezing may comprisestorage in a cryopreservation medium such as DMSO, glycerol, sericin,sugars, or mixtures thereof. Freezing may comprise, for example,incubating the loaded cells at about 4° C. for about 30 minutes to about60 minutes, and then incubating at about −80° C. or colder until use.The loaded cells may then be thawed for use.

Optionally nanoparticle-loaded carrier cells have a viability of aboutat least 20%, 30%, 40%, 50%, 60%, 70% or 80% after one freeze-thawcycle.

Optionally nanoparticle-loaded carrier cells have a viability of atleast about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,or about 80% after about 24 hours of hypothermic storage.

The presently described technology and its advantages will be betterunderstood by reference to the following examples. These examples areprovided to describe specific embodiments of the present technology. Byproviding these specific examples, it is not intended limit the scopeand spirit of the present technology. It will be understood by thoseskilled in the art that the full scope of the presently describedtechnology encompasses the subject matter defined by the claimsappending this specification, and any alterations, modifications, orequivalents of those claims.

EXAMPLES Example 1 Cell Preparation

hMSCs were obtained from adult, healthy BM donors between the ages of 18and 30 years using methods as generally described in U.S. Pat. No.6,355,239. Approximately 60,000 hMSCs were seeded per 24-well in DMEMsupplemented with 10% FBS and 2 mM GlutaMAX™-I (100×, 35050-061,Invitrogen, Carlsbad, Calif.) and cultured in a tissue incubator at 37°C. and 5% CO₂. Cells were cultured for 7 hours at 37° C. and 5% CO2,then the medium was carefully replaced with serum-free medium. Afterovernight starvation, cells were designated for transfection.

Example 2 Protein Transfection of hMSCs with BSA Alexa Fluor® 488Conjugate Using a Cationic Lipid

Approximately 60,000 hMSCs were seeded per 24-well in DMEM supplementedwith 10% FBS and 2 mM GlutaMAX™-I (100×, 35050-061, Invitrogen,Carlsbad, Calif.) and cultured in a tissue incubator at 37° C. and 5%CO₂. After overnight incubation, the medium was replaced by serum-freemedium. After another overnight culture, cells were transfected with BSAAlexa Fluor® 488 conjugate (A13100, Invitrogen, Carlsbad, Calif.).

BSA Alexa Fluor® 488 conjugate was dissolved in PBS containing 2 mMsodium azide to final concentration of 10 μg Alexa Fluor® 488conjugate/μl. QuikEase™ tubes of the BioPORTER® Protein Delivery ReagentQuikEase™ Kit (100077-328, Genesee) were hydrated with 40 μl proteinsolution containing 10 μg (results not shown) or 100 μg BSA Alexa Fluor®488 conjugate, and incubated for about 5 minutes at room temperature.The volume of BioPORTER®/protein mixture was vortexed gently for 3 to 5seconds, and brought to a volume of 0.5 ml with serum-free medium. Thecells were washed to remove all traces of serum prior to transfectionand covered with 125 μl serum-free medium per well. 125 μlBioPORTER®/protein mix was then transferred to the cells, and cells wereincubated for 4 hours (results not shown) or 24 hours. After 4 hours ofincubation, cells were either washed and analyzed, or incubation wascontinued for 20 hours after the addition of 1 volume 20%serum-containing medium for a total incubation time of 24 hours. Cellswere washed with serum-free medium and analyzed by fluorescencemicroscopy and FACS analysis. Results are shown in FIG. 16. Panel a)fluorescence microscopy of hMSCs transfected with BSA Alexa Fluor® 488conjugate using BioPORTER® QuikEase™ Reagent (Genesee and d) FACSanalysis of hMSCs transfected with BSA Alexa Fluor® 488 conjugate usingBioPORTER® QuikEase™ Reagent (Genesee).

This example demonstrates specific examples of generic techniques usefulfor loading carrier cells according to the present invention.

Example 3 Protein Transfection of hMSCs with BSA Alexa Fluor® 488Conjugate Using a Lipid Transfection Reagent

hMSCs were prepared as described in Example 2. Lipodin-Pro™ ProteinTransfection Reagents (product no. 500100, Abbiotech, LLC, San Diego,Calif.) were allowed to equilibrate at room temperature and vortexed for10 seconds at highest setting before use. 2 μl LipodinPro™ reagent wastransferred to a sterile 1.5 ml microcentrifuge tube. 10 μl proteinsolution containing 10 μg (results not shown) or 100 μg BSA Alexa Fluor®488 conjugate were added to the tube. The reaction was incubated for 15minutes at room temperature. The cells were washed to remove all tracesof serum prior to transfection and covered with 390 μl serum-free mediumper well. 100 μl serum-free medium was added to the reaction, and themixture transferred to the cell culture well. Cells were incubated at37° C. and 5% CO2. After 4 hours of incubation, cells were either washedand analyzed (results not shown), or 1 volume 20% serum-containingmedium was added to the cells and incubation continued for 20 hours fora total incubation time of 24 hours. Cells were washed with PBS andanalyzed by fluorescence microscopy and FACS analysis. Results are shownin FIG. 16. Panel: b) fluorescence microscopy of hMSCs transfected withBSA Alexa Fluor® 488 conjugate using Lipodin-Pro™ Transfection Reagents(Abbiotech); and Panel e) Fluorescent FACS analysis of hMSCs transfectedwith BSA Alexa Fluor® 488 conjugate using Lipodin-Pro™ TransfectionReagents (Abbiotech).

This example demonstrates specific examples of generic techniques usefulfor loading carrier cells according to the present invention.

Example 4 Protein Transfection of hMSCs with BSA Alexa Fluor® 488Conjugate Using a Cationic Amphiphile

hMSCs were prepared as described in Example 2. A 5 μg/μl BSA AlexaFluor® 488 conjugate solution was prepared. 10 μg (results not shown) or100 μg BSA were transferred to an eppendorf tubes, and 20 mM Hepessolution added for a final volume of 100 μl per tube. 4 μl PULSin™Delivery Reagent (Genesee Scientific, San Diego, Calif.) was added tothe tubes. The tubes were vortexed and briefly spun down. The reactionwas incubated for 15 minutes at room temperature. After the cells werewashed to remove all traces of serum, 900 μl serum-free medium and 100μl PULSin™/protein mix were added to the cells. The cells were incubatedfor 4 hours at 37° C. in 5% CO2 in a tissue culture incubator. After the4 hour incubation, cells were either washed and analyzed (results notshown), or 1 volume 20% serum-containing medium was added to the welland the incubation continued for 20 hours for a total incubation time of24 hours. Before analysis with a fluorescence microscope and FACSanalysis, cells were washed with PBS. Results are shown in FIG. 16,Panel c) fluorescence microscopy of hMSCs transfected with BSA AlexaFluor® 488 conjugate using PULSin™ Delivery Reagent (GeneseeScientific); Panel f) FACS analysis of hMSCs transfected with BSA AlexaFluor® 488 conjugate using PULSin™ Delivery Reagent (GeneseeScientific).

This example demonstrates specific examples of generic techniques usefulfor loading carrier cells according to the present invention.

Example 5 Cell Loading Using a Cationic Lipid

hMSCs were prepared as described in Example 1. QuikEase™ tubes of thecationic lipid BioPORTER® Protein Delivery Reagent QuikEase™ Kitcontaining the dried BioPORTER® reagent were hydrated with 100 μl AlexaFluor®-488-FluoroNanogold™-anti-mouse Fab′ (product no. 7202,Nanoprobes, Yaphank, N.Y.), 1.4 nm gold particles attached toaffinity-purified Fab′ fragment. The covalently attached fluorophoreAlexa Fluor®-488, enables detection of the gold nanoparticles byfluorescence microscopy. The goat anti-mouse Fab′ attached to thesenanogold particles had no particular functionality in the experiments,but could be used as a secondary reagent to detect primary mouseantibodies interacting with a marker expressed by cells. The reactionwas incubated for 5 minutes at room temperature. The final volume of theBioPORTER®/protein mixture was vortexed gently for 3 to 5 seconds, andbrought to a volume of 0.5 ml with serum-free medium.

The cells were washed to remove all traces of serum prior totransfection and covered with 125 μl serum-free medium per well. 125 μlBioPORTER®/nanogold mix were transferred to the cells to reach a finalconcentration of 1:10 FluoroNanogold™. After 4 hours of incubation,cells were either washed and analyzed (results not shown), or incubationwas continued for 20 hours after the addition of 1 volume of 20%serum-containing medium for a total incubation time of 24 hours.

Cells were washed with serum-free medium and analyzed by fluorescencemicroscopy and FACS. hMSCs with and without FluoroNanogold™, but noprotein transfection reagent, were used as negative controls. hMSCsmodified with Alexa-BSA were used as positive control. The results areshown in FIG. 4. As seen in FIG. 4, nanoparticles can be successfullyloaded into MSCs, and are thereby useful as therapeutic or imagingcontrast agents.

Example 6 Cell Loading Using a Lipid Transfection Reagent

hMSCs were prepared as described in Example 1. The lipid transfectionreagent Lipodin-Pro™ and protein transfection reagents were allowed toequilibrate at room temperature and vortexed for 10 seconds at highestsetting before use. 2 μl LipodinPro™ reagent was transferred to asterile 1.5 ml microcentrifuge tube. 10 μl FluoroNanogold™ was added tothe tube and mixed by pipetting. The reaction was incubated for 15minutes at room temperature.

The cells were washed to remove all traces of serum prior totransfection and covered with 390 μl serum-free medium per well. 100 μlserum-free medium was added to the reaction, and the mixture transferredto the culture well for a final concentration of 1:50 FluoroNanogold™.Cells were incubated at 37° C. and 5% CO2 in a tissue culture incubator.After 3 hours of incubation, cells were either washed and analyzed(results not shown), or 1 volume 20% serum-containing medium was addedto the cells and incubation continued for 20 hours for a totalincubation time of 24 hours.

Cells were washed with PBS and analyzed by fluorescence microscopy andFACS. hMSCs with and without FluoroNanogold™, but no proteintransfection reagent, were used as negative controls. hMSCs modifiedwith Alexa-BSA were used as positive control. Results are shown in FIG.5. As seen in FIG. 5, nanoparticles can be successfully loaded intoMSCs, and are thereby useful as therapeutic or imaging contrast agents.

Example 7 Cell Loading Using a Cationic Amphiphile

hMSCs were prepared as described in Example 1. 100 μl FluoroNanogold™was transferred to an eppendorf tube. 4 μl of the cationic amphiphilePULSin™ Delivery Reagent was added to the tube, and the tube wasvortexed and briefly spun down. The reaction was incubated for 15minutes at room temperature.

After the cells were washed to remove all traces of serum, 900 μlserum-free medium and 100 μl PULSin™/nanogold mix were added to thecells for a final concentration of 1:10 FluoroNanogold™. The cells wereincubated for 4 hours at 37° C. in 5% CO2 in a tissue culture incubator.After the 4 hour incubation, cells were either washed and analyzed(results not shown), or 1 volume 20% serum-containing medium was addedto the well and the incubation continued for 20 hours for a totalincubation time of 24 hours.

Before analysis with a fluorescence microscope, cells were washed withPBS. hMSCs with and without FluoroNanogold™, but no protein transfectionreagent, were used as negative controls. hMSCs modified with Alexa-BSAwere used as positive control. Results are shown in FIG. 6. As seen inFIG. 6, nanoparticles can be successfully loaded into MSCs, and arethereby useful as therapeutic or imaging contrast agents.

Example 8 Cell Loading Using a Cationic Peptide

hMSCs were prepared as described in Example 1. hMSCs were modified usingprotamine sulfate as FluoroNanogold™ carrier. Prior to transfection, aprotamine sulfate stock solution of 10 mg/ml of the cationic peptideprotamine sulfate (product no. P4020, Sigma-Aldrich, Allentown, Pa.) wasprepared.

Different concentrations of protamine sulfate and FluoroNanogold™ weretested. FluoroNanogold™ concentrations ranged from 1:1 to 1:5, protaminesulfate concentrations ranged from 5 μg/ml to 50 μg/ml (see Table 1).Negative controls were either prepared without protamine sulfate orwithout FluoroNanogold™. Also unmodified MSCs were used as additionalnegative controls. hMSCs modified with Alexa-BSA were used as positivecontrol. Cells were washed with PBS and analyzed by fluorescencemicroscopy and FACS. The highest loading was achieved with 1:1FluoroNanogold™, and 50 μg/ml protamine sulfate. Results are shown inFIG. 7. As seen in FIG. 7, nanoparticles can be successfully loaded intoMSCs, and are thereby useful as therapeutic or imaging contrast agents.

TABLE 1 Transfection Conditions 1:4 1:3 1:2 1:1 FluoroNanogold ™FluoroNanogold ™ FluoroNanogold ™ FluoroNanogold ™ 50 μg/ml Protamine 50μg/ml Protamine 50 μg/ml Protamine 50 μg/ml Protamine 1:4 1:3 1:2 1:1FluoroNanogold ™ FluoroNanogold ™ FluoroNanogold ™ FluoroNanogold ™ 5μg/ml Protamine 5 μg/ml Protamine 5 μg/ml Protamine 5 μg/ml Protamine1:5 1:5 1:5 1:5 FluoroNanogold ™ FluoroNanogold ™ FluoroNanogold ™FluoroNanogold ™ 5 μg/ml Protamine 10 μg/ml Protamine 30 μg/ml Protamine50 μg/ml Protamine

Example 9 Cell Loading Using a Cationic Peptide and a Cationic LipidTransfection Agent

hMSCs were prepared as described in Example 1. Prior to transfection, aprotamine sulfate stock solution of 10 mg/ml protamine sulfate (productno. P4020, Sigma-Aldrich, Allentown, Pa.) was prepared. QuikEase™ tubesof the BioPORTER® Protein Delivery Reagent QuikEase™ Kit containing thedried BioPORTER® reagent were hydrated with 100 μl FluoroNanogold™. Insamples using protamine sulfate, 5 ug protamine sulfate was added. Thereaction was incubated for 5 minutes at room temperature, then vortexedgently for 3 to 5 seconds. The final volume of theBioPORTER®/nanogold/protamine sulfate mixture was then brought to 500 μlwith serum-free medium.

The cells were washed to remove all traces of serum prior totransfection and covered with 125 μl serum-free medium per well. 125 μlof the BioPORTER®/nanogold/protamine sulfate mix was transferred to thecells to reach a final concentration of 1:10 FluoroNanogold™, and 5μg/ml protamine sulfate in the cell suspension. After 4 hours ofincubation, cells were either washed and analyzed (results not shown),or incubation was continued for 20 hours after the addition of 1 volume20% serum-containing medium (total incubation time of 24 hours).

Cells were washed with serum-free medium and analyzed by fluorescencemicroscopy and FACS. Unmodified hMSCs and hMSCs modified withFluoroNanogold™, but no transfection reagent, were used as negativecontrols. hMSCs modified with Alexa-BSA were used as positive control.Results are shown in FIG. 8. As seen in FIG. 8, nanoparticles can besuccessfully loaded into MSCs, and are thereby useful as therapeutic orimaging contrast agents.

Example 10 Cell Loading Using a Cationic Peptide and a LipidTransfection Reagent

hMSCs were prepared as described in Example 1. Prior to transfection, aprotamine sulfate stock solution of 10 mg/ml protamine sulfate (productno. P4020, Sigma-Aldrich, Allentown, Pa.) was prepared. Lipodin-Pro™Protein Transfection Reagents were allowed to equilibrate at roomtemperature and were vortexed for 10 seconds at the highest settingbefore use. 2 μl LipodinPro™ reagent was transferred to a sterile 1.5 mlmicrocentrifuge tube. 10 μl FluoroNanogold™ pre-mixed with 2.5 μgprotamine solution were added to the tube and mixed by pipetting. Thereaction was incubated for 15 minutes at room temperature.

The cells were washed to remove all traces of serum prior totransfection and covered with 390 μl serum-free medium per well. 100 μlserum-free medium were added to the reaction, and the mixturetransferred to the culture dish for a final concentration of 1:50FluoroNanogold™. Cells were incubated at 37° C. in a tissue cultureincubator.

After 4 hours of incubation, cells were either washed and analyzed(results not shown), or 1 volume 20% serum-containing medium was addedto the cells and incubation continued for 20 hours (total incubationtime of 24 hours). Cells were washed with PBS and analyzed byfluorescence microscopy and FACS. Unmodified hMSCs and hMSCs modifiedwith FluoroNanogold™, but no transfection reagent, were used as negativecontrols. hMSCs modified with Alexa-BSA were used as positive control.

Results are shown in FIG. 9. As can be seen, nanoparticles can besuccessfully loaded into MSCs, and are thereby useful as therapeutic orimaging contrast agents.

Example 11 Cell Loading Using a Cationic Peptide and a CationicAmphiphile

hMSCs were prepared as described in Example 1. Prior to transfection, aprotamine sulfate stock solution of 10 mg/ml protamine sulfate (productno. P4020, Sigma-Aldrich, Allentown, Pa.) was prepared. 100 μlFluoroNanogold™ pre-mixed with 5 μg protamine solution was transferredto an eppendorf tube. 4 μl PULSin™ Delivery Reagent was then added tothe tube, and the tube vortexed and briefly spun down. The reaction wasincubated for 15 minutes at room temperature.

After the cells were washed to remove all traces of serum, 900 μl ofculture medium without serum and 100 μl of PULSin™/nanogold/protaminesulfate mix were added to the cells for a final concentration of 1:10FluoroNanogold™ and 5 μg/ml protamine sulfate. The cells were incubatedfor 4 hours at 37° C. and 5% CO2 in a tissue culture incubator.

After the 4 hour incubation, cells were either washed and analyzed(results not shown), or 20% serum-containing medium was added to thewell and the incubation continued for 20 hours (total incubation time of24 hours). Before analysis with a fluorescence microscope and by FACS,cells were washed with PBS. Unmodified hMSCs and hMSCs modified withFluoroNanogold™, but no transfection reagent, were used as negativecontrols. hMSCs modified with Alexa-BSA were used as positive control.As seen in FIG. 10, nanoparticles can be successfully loaded into MSCs,and are useful as therapeutic or imaging contrast agents.

Example 12 Cell Loading Using a Cell Penetrating Peptide and a CationicPeptide Covalently Attached to Au

60,000 hMSCs were seeded in 24-wells and cultured in DMEM supplementedwith 10% FBS and 2 mM GlutaMAX™-I (100×, product no. 35050-061,Invitrogen, Carlsbad, Calif.). Cells were cultured for 5 hours at 37° C.and 5% CO2, before the medium was carefully replaced by serum-freemedium. After overnight starvation, cells were designated fortransfection with the cell penetrating peptide (Arg)₉ gold,H-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-OH (Arg)9 conjugated to 5 nmunlabeled gold nanoparticles (Nanopartz™).

Prior to transfection, 40 mg gold powder was dissolved in 200 μl PBS(200 μg/μl). A protamine sulfate stock solution of 10 mg/ml protaminesulfate (product no. P4020, Sigma-Aldrich, Allentown, Pa.) was made. 300μl samples containing 50 μg/ml protamine sulfate and 100 μg, 200 μg, or300 μg (Arg)9 gold were prepared. An additional sample with increasedprotamine concentration of 100 μg/ml protamine sulfate and 300 μg (Arg)9gold was made. The mix was incubated for 10 minutes at room temperature.

After the cells were washed with serum-free medium, the prepared mix wasadded to the cells. The cells were incubated for 4 hours at 37° C. in a5% CO2 tissue culture incubator. After the 4 hour incubation, 1 volumeof 20% serum-containing medium was added to the well and the incubationcontinued for 20 hours (total incubation time of 24 hours). UnmodifiedhMSCs, and hMSCs modified with (Arg)9 gold without carrier were used asnegative controls. hMSCs modified with FluoroNanogold™ were used aspositive control. After incubation, positive controls were analyzed byfluorescence microscopy.

Cells were washed with PBS and a selection of samples was analyzed byneutron activation (BioPal™, Worcester, Mass.) to determine the goldcontent of each sample. The results are shown in Table 2. As seen inTable 2, nanoparticles can be successfully loaded into MSCs, and arethereby useful as therapeutic or imaging contrast agents.

TABLE 2 Uptake of Nanoparticles Protamine Condition (Arg)9 sulfate %Gold uptake (concentrations) concentration concentration in Cellsgold_(hi) carrier_(hi) 1.0 μg/μl 100 μg/ml  12.6% gold_(hi) carrier_(lo)1.0 μg/μl 50 μg/ml 11.5% gold_(med) carrier_(lo) 0.7 μg/μl 50 μg/ml13.7% gold_(lo) carrier_(lo) 0.3 μg/μl 50 μg/ml 6.6% gold_(hi) 1.0 μg/μl— 0.2% gold_(lo) 0.3 μg/μl — 0.1%

Table 2 Legend: (Arg)9gold concentrations: Gold lo=100 μg (Arg)9gold,Gold med=200 μg (Arg)9gold, Gold hi=300 μg (Arg)₉gold. Carrier lo=50μg/ml protamine sulfate, carrier hi=100 μg/ml protamine sulfate. Finalvolume=300 μl per sample.

Example 13 Cell Loading with 1.9 nm Gold Nanoparticles Using a CationicPeptide

60,000 hMSCs were seeded in 24-wells and cultured in Dulbecco's ModifiedEagle Medium (DMEM) supplemented with 10% FBS and 2 mM GlutaMAX™-I(100×, product no. 35050-061, Invitrogen, Carlsbad, Calif.). Cells werecultured for 5 hours at 37° C. and 5% CO₂, before the medium wascarefully replaced by serum-free medium. After overnight starvation,cells were transfected with AuroVist™, unlabeled 1.9 nm goldnanoparticles (Nanoprobes). Aurovist™ is a commercially availablepreparation of 1.9 nm gold nanoparticles.

Prior to transfection, 40 mg gold powder was dissolved in 200 μl PBS(200 μg/μl). A protamine sulfate stock solution of 10 mg/ml protaminesulfate (product no. P4020, Sigma-Aldrich, Allentown, Pa.) was made. 300μl samples containing 50 μg/ml protamine sulfate and 100 μg, 500 μg, or1 mg Aurovist™ were prepared. An additional sample with increasedprotamine concentration of about 100 μg/ml protamine sulfate and 1 mgAurovist™ was made. The mix was incubated for 10 minutes at roomtemperature. After the cells were washed with serum-free medium, theprepared mix was added to the cells. The cells were incubated for 4hours at 37° C. and 5% CO2 in a tissue culture incubator. After the 4hour incubation, about 1 volume of 20% serum-containing medium was addedto the well and the incubation continued for 20 hours (total incubationtime of 24 hours). Unmodified hMSCs, and hMSCs modified with (Arg)9 goldwithout carrier were used as negative controls. hMSCs modified withFluoroNanogold™ were used as positive control. After incubation,positive controls were analyzed by fluorescence microscopy.

Cells were washed with PBS and a selection of samples was analyzed byneutron activation (BioPal™) to determine the gold content of eachsample. The results are shown in Table 3. As seen in Table 3,nanoparticles can be successfully loaded into MSCs, and are therebyuseful as therapeutic or imaging contrast agents.

TABLE 3 Uptake of Nanoparticles Protamine % Gold Condition Aurovist ™sulfate uptake in (concentrations) concentration concentration Cellsgold_(hi) carrier_(hi) 3.3 μg/μl 100 μg/ml  2.1% gold_(hi) carrier_(lo)3.3 μg/μl 50 μg/ml 1.6% gold_(med) carrier_(lo) 1.7 μg/μl 50 μg/ml 1.5%gold_(lo) carrier_(lo) 0.3 μg/μl 50 μg/ml 0.7% gold_(hi) 3.3 μg/μl —0.1% gold_(lo) 0.3 μg/μl — 0.0%

Table 3 Legend: Transfection of hMSCs with different concentrations ofAurovist™ and protamine sulfate as carrier. Aurovist™ concentrations:Gold_(lo)=100 μg Aurovist™, Gold_(med)=500 μg Aurovist™, Gold_(hi)=1 mgAurovist™. Carrier_(lo)=50 μg/ml protamine sulfate, carrier_(hi)=100μg/ml protamine sulfate. Final volume=300 μl per sample.

Example 14 Large Scale Cell Loading with 1.9 nm Gold Nanoparticles Usinga Cationic Peptide (6-well)

288,000 hMSCs were seeded in 6 wells and cultured in DMEM supplementedwith 10% FBS and about 2 mM GlutaMAX™-I (100×, product no. 35050-061,Invitrogen, Carlsbad, Calif.). Cells were cultured for 6 hours at 37° C.and 5% CO2, before the medium was carefully replaced by serum-freemedium. After overnight starvation, cells were transfected withAuroVist™, unlabeled 1.9 nm gold nanoparticles (Nanoprobes).

Prior to transfection, 40 mg gold powder was dissolved in 200 μl PBS(200 μg/μl). A protamine sulfate stock solution of 10 mg/ml protaminesulfate (product no. P4020, Sigma-Aldrich, Allentown, Pa.) was made.1440 μl samples containing 20 μg/ml or 50 μg/ml protamine sulfate, and50 μg/cm² or 100 μg/cm² Aurovist™ were prepared. The mix was incubatedfor 10 minutes at room temperature. After the cells were washed withserum-free medium, the prepared mix was added to the cells. The cellswere incubated for 4 hours at 37° C. in 5% CO₂ in a tissue cultureincubator. After the 4 hour incubation, 1 volume of 20% serum-containingmedium was added to the well and the incubation continued for 20 hours(total incubation time of 24 hours).

Chemotaxis assays were performed to test the migration ability of hMSCsafter modification with gold nanoparticles. Unmodified hMSCs, hMSCsmodified with Aurovist™ without carrier, and hMSCs exposed to protaminesulfate only were used as negative controls. After overnight culture,cells were washed with serum-free medium and starved for 1 hour inserum-free medium. After starvation, cells were washed with PBS andharvested with 0.125% trypsin without ethylenediaminetetraacetic acid(EDTA). Cells were resuspended in DMEM, cell number and viability wasanalyzed by hematocytometer (Gold 19, 20, 21). DMEM was added to thecells to reach a final concentration of 0.5×10⁶ cells/ml. 500 μl DMEM,or 500 μl DMEM supplemented with 30% FBS and 2 mM GlutaMAX™-I was addedto the lower chamber of a 24-well culture dish. 50,000nanoparticle-loaded cells or unmodified MSCs (100 μl cell suspension)were transferred to transwell inserts with 8 um pore size (Corning, No.3422), and the inserts placed into the prepared wells. The cells wereincubated for 21 hours at 37° C. in a 5% CO2 tissue culture incubator.After incubation, cells were removed from the upper side of the insert.Cells on the lower side of the insert were stained for 18 minutes in an0.2% Gentian violet solution (in 4% paraformaldehyde (PFA)). The insertswere washed and residual cells were removed from the upper side of thefilter. The filters were placed in a 24-well culture dish prepared with500 μl PBS per well and the migrated cells analyzed with LM microscopyat 40× magnification.

Cell viability results are shown in Table 4. The table indicates thenumber of alive (viable) hMSCs after transfection and isolation of thecells, and the percentage of alive hMSCs after transfection and cellisolation compared to the number of seeded cells, as well as compared tothe number of harvested cells of the negative control. As seen in Table4, MSCs can be loaded according to the present invention on a largescale and exhibit substantial viability, and are useful as therapeuticand imaging contrast agents.

TABLE 4 Viability of cells after 24 hour transfection with goldnanoparticles % of Neg % of Seeded Ctrl No. Cells Cells (145000Description Alive (288000 cells) cells) hMSCs (Neg Ctrl) 145,000 50%100% No gold, 50 μg/ml Prot 116,000 40%  80% 50 ug/cm² Aurovist ™,107,000 37%  74% 50 μg/ml Prot 50 ug/cm² Aurovist ™, 116,600 40%  80% 20μg/ml Prot 100 ug/cm² Aurovist ™, 152,000 53% 105% 50 μg/ml Prot 100ug/cm² Aurovist ™, 246,000 85% 170% 20 μg/ml Prot 50 ug/cm² Aurovist ™174,000 60% 120% 100 ug/cm² Aurovist ™ 111,000 39%  77%

Results from the chemotaxis assay are shown in FIG. 11 and FIG. 12. FIG.11 a depicts 10× magnification of unmodified hMSCs (positive control).FIG. 11 b depicts 10× magnification of hMSCs loaded with goldnanoparticles. FIG. 12 depicts 20× magnification of hMSCs loaded withgold nanoparticles. Gold nanoparticles are clearly visible in thecytoplasm of MSCs in FIG. 12. As seen in FIG. 11, hMSCs with goldnanoparticles in the cytoplasm retained their migration ability.

Example 15 Large Scale Cell Loading with Gold Nanoparticles Using aCationic Peptide (T80 Flask)

Approximately 2.4 million hMSCs were seeded in a T80 flask and culturedin DMEM supplemented with 10% FBS and 2 mM GlutaMAX™-I (100×, productno. 35050-061, Invitrogen, Carlsbad, Calif.). Cells were cultured for 6hours at 37° C. and 5% CO2, before the medium was carefully replacedwith serum-free medium. After overnight starvation, cells weretransfected with AuroVist™, unlabeled 1.9 nm gold nanoparticles(Nanoprobes).

Prior to transfection, 40 mg gold powder was dissolved in 200 μl PBS(200 μg/μl). A protamine sulfate stock solution of 10 mg/ml protaminesulfate (product no. P4020, Sigma-Aldrich, Allentown, Pa.) was made. 12ml samples containing 20 μg/ml or 50 μg/ml protamine sulfate, and 50μg/cm2 Aurovist™ were prepared. The mix was incubated for 10 minutes atroom temperature. After the cells were washed with serum-free medium,the prepared mix was added to the cells. The cells were incubated for 4hours at 37° C. in a 5% CO2 tissue culture incubator. After the 4 hourincubation, 1 volume of 20% serum-containing medium was added to thewell and the incubation continued for 20 hours (total incubation time of24 hours). Cells were washed with PBS. Cell viability was analyzed withtrypan blue. The results are shown in Table 5. As seen in Table 5, MSCscan be loaded according to the present invention on a large scale,exhibit substantial viability, and are useful as therapeutic and imagingcontrast agents.

TABLE 5 Viability of cells after 24 hour transfection with goldnanoparticles % of Seeded Cells % of Neg Ctrl No. Cells (2.4 million(1.1 million Description alive cells) cells) hMSCs (Neg Ctrl) 1,125,00047% 100%  50 ug/cm² Aurovist ™, 862,500 36% 77% 50 μg/ml Prot 50 ug/cm²Aurovist ™, 912,500 38% 81% 20 μg/ml Prot

To test cell proliferation, 125,000 nanoparticle-loaded cells werereplated in a T80 flask and cultured at 37° C. in 5% CO2 for 5 days.Replated cell proliferated and reached confluency after 5 days.Unmodified hMSCs were used as negative control. Nanoparticle-loadedcells retain the ability to proliferate comparable to unmodified MSCs(data not shown).

Example 16 Large Scale Cell Loading with Gold Nanoparticles Using aCationic Peptide (T185 Flask)

Approximately 5.5 million hMSCs were seeded in a T185 flask and culturedin DMEM supplemented with 10% FBS and 2 mM GlutaMAX™-I (100×, productno. 35050-061, Invitrogen, Carlsbad, Calif.). Cells were cultured for 6hours at 37° C. and 5% CO2, before the medium was carefully replaced byserum-free medium. After overnight starvation, cells were transfectedwith AuroVist™, unlabeled 1.9 nm gold nanoparticles (Nanoprobes).

Prior to transfection, 40 mg gold powder was dissolved in 200 μl PBS(200 μg/μl). A protamine sulfate stock solution of 10 mg/ml protaminesulfate (product no. P4020, Sigma-Aldrich, Allentown, Pa.) was made. 28ml samples containing 20 μg/ml or 50 μg/ml protamine sulfate, and 50μg/cm2 or 100 μg/cm2 Aurovist™ were prepared. The mix was incubated for10 minutes at room temperature. After the cells were washed withserum-free medium, the prepared mix was added to the cells. The cellswere incubated for 4 hours at 37° C. in a 5% CO2 tissue cultureincubator. After the 4 hour incubation, 1 volume of 20% serum-containingmedium was added to the well and the incubation continued for 20 hours(total incubation time of 24 hours). Cells were washed with PBS. Cellviability was analyzed with trypan blue. Results are shown in Table 6.Unmodified hMSCs were used as negative control. As seen in Table 6,these cells can be loaded on a large scale with sufficient viability foruse in therapy and imaging.

TABLE 6 Cell Viability of gold nanoparticle-loaded cells % of SeededCells % of Neg Ctrl No. Cells (5.55 million (3.94 million DescriptionAlive cells/flask) cells/flask) hMSCs (Neg Ctrl) 3.94 million 71% 100% 50 μg/cm² Aurovist ™, 3.16 million 57% 80% 50 μg/ml Prot 50 μg/cm²Aurovist ™, 2.98 million 54% 76% 20 μg/ml Prot 100 μg/cm² Aurovist ™,3.64 million 66% 92% 20 μg/ml Prot

After cell isolation, 1 million nanoparticle-loaded cells were replatedper T80 vial and cultured in a tissue incubator at 37° C. in 5% CO2.After 4 hours, the medium was changed to remove any remaining free goldnanoparticles. The cells were then incubated overnight at 37° C. and 5%CO2. The next day, the cells were washed once with DMEM supplementedwith 10% FBS and 2 mM GlutaMAX™-I, washed twice with PBS, and harvestedwith 0.05% trypsin. The cells were resuspended in DMEM supplemented with10% FBS and 2 mM GlutaMAX™-I and counted, The cell proliferation resultsare shown in FIG. 13. Upper left: Unmodified hMSCs. Upper right: hMSCsloaded with 50 μg/cm2 Aurovist™, 50 μg/ml protamine sulfate. Lower left:hMSCs modified with 50 μg/cm2 Aurovist™, 20 μg/ml protamine sulfate.Lower right: hMSCs modified with 100 μg/cm2 Aurovist™, 20 μg/mlprotamine sulfate.

Nanoparticle-loaded MSCs show population density comparable tounmodified hMSCs. Taken together, these results demonstrate a largescale preparation of gold nanoparticle-loaded MSCs which retainviability and remarkable proliferative activity.

Example 17 Transfection with Gold Nanoparticles Using a Cationic Peptideand Washing

hMSCs were prepared and modified as described in Example 16. After 24hours of incubation with Aurovist™, cells were washed three times withDMEM supplemented with 10% FBS and 2 mM GlutaMAX™-I, followed by anincubation of 30 minutes to 1 hour at 37° C., and 5% CO2 in a tissueculture incubator. The cells were then washed twice with PBS andharvested.

The CYTOMATE® cell processing system is a CE-marked, automated,functionally closed system cell washer with the flexibility to processsmall to large volumes of white cell products and the capability to actas a fluid transfer device. The underlying technology of the CYTOMATE®cell processing system is a spinning membrane with a defined pore size,which ensures cell filtration against a counter-flow buffer circulationand is connected to different bags in a functionally closed system. Toremove free gold particles from the cell suspension, isolatednanoparticle-loaded cells were 1) washed in the CYTOMATE® cellprocessing system (Fenwal™, Lake Zurich, Ill.), or 2) replated in cellculture flasks (1 million cells per T80 flask) and cultured overnight at37° C. in 5% CO2 and DMEM supplemented with 10% FBS and 2 mMGlutaMAX™-I.

15 million cells in a volume of 100 ml were washed in the CYTOMATE®.After the wash, 83% of the nanoparticle-loaded cells could be recoveredwhich is comparable to the recovery of unmodified hMSCs. FIG. 14 depictsan overview of the described nanoparticle-loaded cell generationprocess.

The gold content of 1 million and 2 million nanoparticle loaded cells ofeach group was analyzed by neutron activation. Table 7 shows goldcontent of nanoparticle-loaded cells after isolation and washing orreplating. Results show that cell washing with the CYTOMATE® removed asubstantial amount of free gold nanoparticles from the suspension ofisolated gold-loaded cells, which could not be removed by replating ofthe cells.

Results also show, the loading of 2 million hMSCs with 1088 μgAurovist™.

TABLE 7 Gold content of nanoparticle-loaded MSCs (in ug) 50 μg/cm² 100g/cm² Aurovist ™ used for Aurovist ™ used for transfection transfectionCells washed, 1 million 356 468 Cells washed, 2 million 674 1,088 Cellsreplated, 1 million 352 842 Cells replated, 2 million 692 2,203

Example 18 Optional Cell Isolation and Washing Method

hMSCs were prepared and modified as described in Example 16. After 24hours of incubation with Aurovist™, cells were washed three times withDMEM supplemented with 10% FBS and 2 mM GlutaMAX™-I, followed by twowashing steps with PBS.

To remove additional gold nanoparticles from the cell culture, 20 mltrypsin were added per flask for 1 to 2 minutes and the flask movedcontinuously. When free nanoparticles were observed in the culture, butthe cells were still attached, the trypsin was collected and discarded.Additional 2 ml trypsin were added to each flask for 6 to 10 minutes andthe cells harvested. The cells were washed with PBS and then filteredthrough a BD FACS filter

After cell isolation, the cell suspension was prepared for injectioninto animals.

Example 19 Large Scale Cell Loading with Gold Nanoparticles Using aCationic Peptide (Cell Factories)

38 million hMSCs were seeded in a cell factory (2 trays) with a surfacearea of 1264 cm² and cultured in DMEM supplemented with 10% fetal bovineserum (FBS) and 2 mM GlutaMAX™-I (100×, product no. 35050-061,Invitrogen, Carlsbad, Calif.). Cells were cultured for 6 hours at 37° C.and 5% CO2, before the medium was carefully replaced by serum-freemedium. After overnight starvation, cells were transfected withAuroVist™, unlabeled 1.9 nm gold nanoparticles (Nanoprobes).

Prior to transfection, 40 mg gold powder was dissolved in 200 μl PBS(200 μg/μl). A protamine sulfate stock solution of 10 mg/ml protaminesulfate (product no. P4020, Sigma-Aldrich, Allentown, Pa.) was made. 189ml samples containing 20 μg/ml protamine sulfate, and 50 μg/cm2Aurovist™ were prepared. The mix was incubated for 10 minutes at roomtemperature. After the cells were washed with serum-free medium, theprepared mix was added to the cells. The cells were incubated for 4hours at 37° C. in a 5% CO2 tissue culture incubator. After the 4 hourincubation, 1 volume of 20% serum-containing medium was added to thewell and the incubation continued for 20 hours (total incubation time of24 hours). After 24 hours of incubation with Aurovist™, cells werewashed three times with DMEM supplemented with 10% FBS and 2 mMGlutaMAX™-I, followed by an incubation of 30 minutes to 1 hour at 37° C.and 5% CO2 in a tissue culture incubator. The cells were then washedtwice with PBS and harvested. After harvest, the isolatednanoparticle-loaded cells were washed in the CYTOMATE® cell processingsystem (Fenwal™).

Generation of nanoparticle-loaded cells using a cell factory with asurface area of 1264 cm2 resulted in an average yield of 22 millionnanoparticle-loaded cells.

Example 20 Cryopreservation of Nanoparticle-Loaded Cells

MSCs were loaded with nanoparticles and isolated as described in Example19. 1 million nanoparticle-loaded cells were centrifuged at 1430 rpm for5 minutes. After discarding the supernatant, the cell pellet wasresuspended in 500 μl cold cryoprotectant and transferred to a cryovial.The cells were kept in a freezing container at −80 C (freezing rate of1° C./minute) overnight, and then transferred cells to liquid nitrogen.For cell viability analysis, cells were thawed, centrifuged at 1430 rpmfor 5 minutes, the supernatant removed, and the pellet resuspended inDMEM supplemented with 10% FBS and 2 mM GlutaMAX™-I. The cells were thencounted with trypan blue. Remaining cells were transferred to a T80flask and cultured overnight in DMEM supplemented with 10% FBS and 2 mMGlutaMAX™-I at 37° C. and 5% CO2 in a tissue culture incubator.

As shown in Table 8, the methods below resulted in a range of usefulcell viabilities.

TABLE 8 Survival of nanoparticle-loaded cells after freeze-thaw cycleViable Cells after No Viable Cells after freezing and cells Storagesolution freezing 1 overnight culture 1M Plasmalyte, 20% 915,000 (92%)365,000 (37%) DMSO, 5% HSA 5M Plasmalyte, 20% 3,450,000 (69%) 2,710,000(54%) DMSO, 5% HSA 1M CryoMaxx SF 895,000 (90%) 665,000 (67%) 5MCryoMaxx SF 4,110,000 (82%) 3,340,000 (67%) 1M Plasmalyte, 10% 985,000(99%) 382,500 (39%) DMSO, 5% HSA 5M Plasmalyte, 10% 4,010,000 (80%)2,720,000 (54%) DMSO, 5% HSA

Example 21 Hypothermic Storage of Nanoparticle-Loaded Cells

MSCs were loaded with nanoparticles and isolated as described in Example19. 1 million Nanoparticle-loaded cells were centrifuged at 1430 rpm for5 minutes. After discarding the supernatant, the cell pellet wasresuspended in 1 ml hypothermic solution and transferred to an eppendorftube. Cells were kept at 2 to 8° C. until they were analyzed. For eachtime point a separate sample was generated and stored to avoid multiplehandling of samples. Due to this procedure, survival curves mightinclude raises and descents. For analysis, cells were snipped and mixedper pipet, then counted with trypan blue.

FIG. 15 shows results of hypothermic storage of gold-nanoparticle loadedMSCs in CoolStar (PAA, Dartmouth, Mass.) and HypoThermosol®(Biolifesolutions, Bothell, Wash.). Storage in CoolStar shows high cellviability for 1 overnight storage. Storage in HypoThermosol® proved tobe of advantage, as cell viability remained at about 70% even after 1week storage.

Example 22 In-Vivo Imaging of Nanoparticle-Loaded Cells in NOD/SCID Mice

Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice(n_(test)=4, test group) are selected as the subject. The subjectsreceive a single tail vein infusion of a composition ofnanoparticle-loaded cells (e.g., MSCs) of the present invention wherethe nanoparticles are 1.9 nm gold particles. The composition contains2×10⁶ of nanoparticle-loaded MSCs in a carrier (e.g., 200 μl PBS) and isadministered one or more times (e.g., every three days for a total of 5injections).

For positive control, NOD/SCID mice (n_(ctrl)=2, control group) receivea single tail vein infusion of the nanoparticles (e.g., 2.7 g) in thecarrier.

The distribution of the nanoparticles is analyzed by (computedtomography) CT scanning or planar imaging at one or more time pointsafter administration (e.g., 1 hour and 24 hours after injection).

A full-body CT scan is performed with monochromatic synchrotron x-raysto detect the injected nanoparticles (Dilmanian et al., 1997).Alternatively, a Lorad Medical Systems mammography unit (Hologic, Inc.,Danbury, Conn.; model XDA101827) is used with 8 mAs exposures (0.4 s at22 kVp) for planar imaging (Hainfeld et al., 2006). Other imagingmethods are also useful.

The results demonstrate the presence of nanoparticle-loaded cells in anon-immunocompetent animal in the blood, followed by blood clearance,distribution to the lung, liver, kidney, spleen, and optionally the bonemarrow, prior to being cleared from the body.

Example 23 In-Vivo Imaging of Nanoparticle-Loaded Cells in NOD/SCID Mice

The protocol of Example 22 is generally performed with the followingaddition:

After imaging is completed, 2 test subjects and 1 control subject aresacrificed. Lung, kidney, spleen, liver, and BM are collected and sentout for neutron activation analysis (Hainfeld et al., 2010). NeutronActivation Analysis (NAA) is a nuclear process used to determine theconcentrations of elements in a sample. The sample is introduced intothe intense radiation field of a nuclear reactor, and bombarded withneutrons, causing the elements to form radioactive isotopes. Theradioactive emissions and radioactive decay paths for each element arewell known. Using this information it is possible to study spectra ofthe emissions of the radioactive sample, and determine theconcentrations of the elements within it.

For the remaining test subjects (n_(test)=2) and control animals(n_(ctrl)=1), imaging of the nanoparticle distribution is performed atday 4 and day 7 by CT scanning or planar imaging. After imaging iscompleted, all remaining animal are sacrificed. Lung, kidney spleen,liver, and bone marrow (BM) are collected and sent out for neutronactivation analysis (Hainfeld et al., 2010).

A diagram of the process is depicted in FIG. 1.

The results show that the concentration of nanoparticle-loaded carriercells is correlated with the intensity of the distribution imaged inExample 22, namely in the blood, followed by blood clearance,distribution to the lung, liver, kidney, spleen, and optionally the bonemarrow, prior to being cleared from the body.

Example 24 In-Vivo CT Imaging of Subcutaneously Injected Nanoparticlesin NOD/SCID Mice

Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice(n_(test)=5, test group) were selected as the subject. The animalsreceived a single subcutaneous injection of 10.0 mg, 3.0 mg, 1.0 mg, 0.5mg, or 0.25 mg Aurovist™, unlabeled 1.9 nm gold nanoparticles.

For negative control, NOD/SCID mice (n_(ctrl)=1, control group) receiveda single subcutaneous injection of PBS.

The distribution of the nanoparticles was analyzed by a FLEXMicroSPECT/CT imaging system (Siemens, Malvern, Pa.). Full-body CT scanwere performed at baseline, 30 to 40 seconds post injection, and 3 to 10minutes post injection to detect the nanoparticles.

Nanoparticle Distribution—The results demonstrate that the imagingthreshold for the FLEX MicroSPECT/CT imaging system is reached at a goldnanoparticle concentration of 0.5 mg. Lower concentrations are notdetectable. Larger concentrations are clearly visible. Nanoparticleswere still detected at the site of injection 3 to 10 minutes aftersubcutaneous injection.

Acute Toxicity—No acute toxicity was observed in animal subcutaneouslyinjected with 0.25 mg to 10.0 mg unlabeled 1.9 nm gold nanoparticles

Example 25 Imaging of Nanoparticle-Loaded Cells in Tumors

Subjects (e.g., NOD/SCID) mice are injected with tumor-forming cells(e.g., 2×10⁶ MDAMB231 cells in 200 μL PBS) (n=4), s.c., into the leftflank with a 29-gauge needle to produce a subcutaneous tumor (Loebingeret al., 2009). Tumors are measured every 3 to 5 days with calipers, andthe volume is calculated as 4/3πr3, where r is the radius.

About 28 days after injection of the MDAMB231 cells, when the tumorsreach a volume of 180 mm³ to 220 mm³ (Karnoub et al., 2007, Loebinger etal., Trail 2009), the animals receive a tail vein infusion of acomposition of nanoparticle-loaded cells (e.g., MSCs) of the presentinvention. The nanoparticles are 1.9 nm gold particles. The compositioncontains 2×10⁶ nanoparticle-loaded MSCs in a carrier (e.g., 200 μl PBS)and is administered one or more times (e.g., every three days for atotal of 5 injections).

Imaging of nanoparticles is performed one or more times (e.g., at 1hour, 24 hours and 48 hours after the first injection, 48 hours afterinfusion 2 to 4, and 1 hour and 24 hours after the last injection).After imaging is completed, the tumor, lung, kidney, spleen, liver, andBM of 2 animals are collected and sent out for neutron activationanalysis (Hainfeld et al., 2010). Imaging of the nanoparticledistribution in the two remaining animals is performed one or more times(e.g., at day 4 and day 7).

The imaging results show a co-localization of the signal from thenanoparticles loaded carrier cells (e.g., MSC cells) with thesubcutaneous tumor tissue in the left flank of the animal.

Example 26 Imaging of Nanoparticle-Loaded Cells in Tumors

The protocol of Example 25 is generally performed with the followingmodifications:

After imaging is completed, all remaining animals are sacrificed. Lung,kidney, spleen, liver, and BM are collected and sent out for neutronactivation analysis (Hainfeld et al., 2010). A diagram of the process isdepicted in FIG. 2.

These results show that the concentration of nanoparticle-loaded carriercells is consistent with the distribution imaged in Example 25. Mostnanoparticles show co-localization with the subcutaneous tumor tissue inthe left flank of the animal.

Example 27 In-Vivo X-Ray Imaging of Subcutaneously InjectedNanoparticles in NOD/SCID Mice

A nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse(n=1) was selected as the subject. The animal received a singlesubcutaneous injection of 1.0 mg Aurovist™, unlabeled 1.9 nm goldnanoparticles.

The distribution of the nanoparticles was analyzed by a Faxitron XrayLLC system. A full-body X ray scan was performed 10 minutes postinjection and at later time-points to detect the nanoparticles.

Nanoparticle Distribution—The results demonstrate that gold nanoparticleconcentrations of 1 mg can be visualized with the Faxitron Xray LLC(Lincolnshire, Ill.) system. Image quality was lower compared to the CTimaging results with the FLEX MicroSPECT/CT imaging system. Goldnanoparticles were present at the site of injection for at least onehour post injection.

Acute Toxicity—No acute toxicity was observed in the animal following asubcutaneous 1.0 mg gold nanoparticle injection.

Alternatively, imaging can be performed using monochromatic synchrotronx-rays (Dilmanian et al., 1997) or a Lorad Medical Systems mammographyunit (Hologic, Inc., Danbury, Conn.; model XDA101827; Hainfeld et al.,2006). Other imaging methods are also useful.

Example 28 In-Vivo CT Imaging of Intravenously Injected Nanoparticles inNOD/SCID Mice

Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (n=3)were selected as the subject. The animals received a single intravenousinfusion of 20.0 mg (high dose) or 1.0 mg (low dose) Aurovist™,unlabeled 1.9 nm gold nanoparticles.

The distribution of the nanoparticles was analyzed by a FLEXMicroSPECT/CT imaging system. Full-body CT scans were performed atbaseline, 3 minutes, 10 minutes, 60 minutes, 1 day, and in one subject 7days after injection.

A diagram of the process is depicted in FIG. 1.

Nanoparticle Distribution—The results show that gold nanoparticles werepresent in the kidney and bladder 3 minutes and 10 minutes after highdose injection. At later time-points gold nanoparticles were notdetected in those organs. Accumulation of gold nanoparticles in otherorgans was not observed in healthy mice.

Acute Toxicity—No animal died after injection of 1.0 mg to 20.0 mg goldnanoparticles per tail vein. Waking up from anesthesia after high doseinjection, some mice showed signs of blindness and signs of increasedsensitivity to touch. Mice recovered from symptoms within one hour.

Cageside Observations—Eyes, snout, feet and tail of the animalsreceiving a high dose of gold nanoparticles became dark immediatelyafter injection due to the presence of gold nanoparticles. The colorchange persisted for several days. Low dose injection of goldnanoparticles did not result in a color change of the animal.

Example 29 In-Vivo Imaging of Intravenously Injected Nanoparticle-LoadedCells in NOD/SCID Mice

Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (n=4)were selected as the subject. The animals received a single tail veininfusion of a composition of nanoparticle-loaded cells (e.g., MSCs) ofthe present invention where the nanoparticles are 1.9 nm gold particles.The composition contained 2×10⁶ of nanoparticle-loaded MSCs in a carrier(e.g., 200 μl PBS) and was administered intravenously.

The distribution of the nanoparticles was analyzed by a FLEXMicroSPECT/CT imaging system. Full-body CT scans were performed atbaseline, 60 minutes, 1 day, and in two subjects 4 days and 7 days postinjection.

A diagram of the process is depicted in FIG. 2.

Nanoparticle Distribution—The results demonstrate thatnanoparticle-loaded cells do not accumulate in any organs of the healthyanimals in large quantities prior to being cleared from the body.

Acute Toxicity—No acute toxicity was observed in animal injected with2×10⁶ of nanoparticle-loaded MSCs per tail vein.

Example 30 Importance of Described Cell Isolation Methods for Reductionof Acute Toxicity of Nanoparticle-Loaded Cells

hMSCs were prepared and modified as described in Example 16. After 24hours of incubation with Aurovist™, cells were washed three times withDMEM supplemented with 10% FBS and 2 mM GlutaMAX™-I, followed by twowashing steps with PBS.

Cells were harvested and prepared for injection without further cellpurification.

Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (n=2)were selected as the subject. The animals received a single tail veininfusion of 2×10⁶ of nanoparticle-loaded MSCs in a carrier (e.g., 200 μlPBS).

Mice treated with 2×10⁶ of the nanoparticle-loaded cells in a carrierdied immediately after injection due to lung embolism resulting fromcell aggregation. The results demonstrate the importance of extensivecell washing procedures as described, for instance, in examples 18 and19 including trypsinizating and filtration, or the use of the CYTOMATE®cell processing system (Fenwal™), the COBE® 2991 Cell Processor (Gambro®BCT, Lakewood, Colo.), the kSep® centrifugation system (kSep® Systems,Durham, N.C.), or any other available cell processing device for thereduction of toxicity of nanoparticle loaded cells due to cellaggregation.

Example 31 Radiotherapy Enhanced with Nanoparticle-Loaded Cells

Subjects (e.g., NOD/SCID mice) are injected with tumor-forming cells(e.g., 2×10⁶ MDAMB231 cells in 200 μL PBS), s.c., into the left flank toproduce a subcutaneous tumor (n_(test)=4; n_(ctrl)=8). Tumors aremeasured every 3 to 5 days with calipers and the tumor volume iscalculated.

About 2 to 4 weeks after injection of the tumor forming cells, when thetumors reach a volume of 100 mm³ to 150 mm³, test animals receive a tailvein infusion of a composition of nanoparticle-loaded cells (e.g., MSCs)of the present invention (n_(test)=4). The nanoparticles are unlabeled1.9 nm gold particles. The composition contains 2×10⁶nanoparticle-loaded MSCs in a carrier (e.g., 200 μl PBS) and isadministered one or more times (e.g., every three days for a total of 3to 5 injections). All other animals receive tail vein infusions of 200μl saline (n_(ctrl)=8).

Imaging of nanoparticles is performed at baseline, and 48 to 72 hoursafter each nanoparticle-loaded cell injection. If nanoparticle-loadedcells can be tracked, additional imaging time points may be added toanalyze nanoparticle clearance.

After the nanoparticle-loaded cells reach the tumor site, a 2.5-cmdiameter region of the flank containing the tumor (n_(test)=4;n_(ctrl)=4) is irradiated with therapeutic radiation (e.g., 250 kVpX-rays), for example, through a Thoreaus-1 filter at 5 Gymin-1 (30 Gytotal) using a clinical Siemens Stabilipan X-ray generator (Hainfeld etal., 2010). 4 of the control mice also receive radiotherapy treatment.The remaining 4 control animals stay untreated (n_(ctrl)=8).

After radiation therapy, the subcutaneous tumors are measured every 3days with calipers for a total of four weeks in all groups(n_(total)=12). Dependent on tumor development, radiation therapy may berepeated. A survival analysis is performed. At 12 weeks after radiationtherapy, tumor volume is measured again.

The imaging results show a co-localization of the signal from thenanoparticles loaded carrier cells (e.g., MSC cells) with thesubcutaneous tumor tissue in the left flank of the animal.

The results demonstrate that compositions of the present invention areuseful for imaging when combined with an imaging dose of radiation and,when combined with radiation therapy, facilitate remarkably enhancedkilling of tumor cells resulting in decrease of tumor size and increasein survival rate. In contrast, in control animals (treated with saline),the tumor continues to grow.

A diagram of the process is depicted in FIG. 3.

Example 32 Radiotherapy Enhanced with Nanoparticle-Loaded Cells

Example 31 is generally performed with the following addition:

The tumor, lung, kidney spleen, liver, and BM are collected and sent outfor neutron activation analysis. Neutron Activation Analysis (NAA) is anuclear process used to determine the concentrations of elements in asample. The sample is introduced into the intense radiation field of anuclear reactor, and bombarded with neutrons, causing the elements toform radioactive isotopes. The radioactive emissions and radioactivedecay paths for each element are well known. Using this information itis possible to study spectra of the emissions of the radioactive sample,and determine the concentrations of the elements within it.

These results show that the enhanced killing effect of the radiation isdue to the compositions of the present invention. Gold-nanoparticleloaded cells at the site of the tumor effectively enhance theradiotherapy effect.

A diagram of the process is depicted in FIG. 3.

Example 33 Isolation of Nanoparticle-Loaded MSCs using COBE® 2991 CellProcessor

The protocol of Example 18 is generally performed with the followingmodifications:

After isolation of nanoparticle-loaded cells, the cells are washed withthe COBE® 2991 Cell Processor (Gambro® BCT), instead of being washed inthe CYTOMATE® cell processing system (Fenwal™).

Example 34 Isolation of Nanoparticle-Loaded MSCs using a Sepax® (BiosafeAmerica)

The protocol of Example 18 is generally performed with the followingmodifications:

After isolation of nanoparticle-loaded cells, the cells are washed withthe Sepax® system (Biosafe America, Houston, Tex.), instead of beingwashed in the CYTOMATE® cell processing system (Fenwal™).

Example 35 Isolation of Nanoparticle-Loaded MSCs Using the kSep®

The protocol of Example 18 is generally performed with the followingmodifications:

After isolation of nanoparticle-loaded cells, the cells are washed withthe kSep® centrifugation system (kSep® Systems), instead of being washedin the CYTOMATE® cell processing system (Fenwal™).

In the present specification, use of the singular includes the pluralexcept where specifically indicated.

The citations provided herein are hereby incorporated by reference forthe cited subject matter.

The presently described technology is now described in such full, clear,concise and exact terms as to enable any person skilled in the art towhich it pertains, to practice the same. It is to be understood that theforegoing describes preferred embodiments of the technology and thatmodifications may be made therein without departing from the spirit orscope of the invention as set forth in the appended claims.

1. A cell comprising one or more nanoparticles wherein the cell is amesenchymal stem cell (MSC) and the one or more nanoparticles comprisesa high-Z element, wherein the high-Z element has an atomic number of atleast 27 and wherein the high-Z element is in a majority amount in theone or more particles by weight.
 2. A cell comprising one or morenanoparticles wherein the cell is a mesenchymal stem cell (MSC) and theone or more nanoparticles comprises a high-Z element, wherein the high-Zelement has an atomic number of at least 27 and wherein the high-Zelement is in a primary image enhancer.
 3. A cell comprising one or morenanoparticles wherein the cell is a mesenchymal stem cell (MSC), whereinthe nanoparticle comprises a core and the core comprises a high-Zmaterial selected from the group consisting of high-Z elements with anatomic number of at least 27, heavy metal oxides, superconductors,paramagnetic materials, and quantum dots.
 4. The cell of claim 3 whereinthe high-Z material comprises gold or iron-oxide.
 5. The cell of claim 4wherein the majority of the one or more nanoparticles has a diameter ina range of about 0.1 nm to about 20 nm.
 6. The cell of claim 5 whereinthe total mass of the one or more nanoparticles is in a range of about0.05 atto grams to about 500 atto grams.
 7. The cell of claim 5 whereinthe one or more nanoparticles is present in a range of about 1 to about10,000 nanoparticles.
 8. The cell of claim 7 wherein the total mass ofthe one or more nanoparticles is in a range of about 0.05 atto grams toabout 500 atto grams.
 9. The cell of claim 5 wherein the high-Z elementis gold.
 10. A composition comprising a plurality of MSC cells whereinat least about 1% of the MSC cells comprise one or more nanoparticles,wherein the nanoparticle comprises a gold or iron-oxide core, andwherein the majority of the one or more nanoparticles has a diameter ina range of about 0.1 nm to about 20 nm.
 11. The composition of claim 10wherein the plurality of MSC cells are present in an amount of at leastabout 100,000 in number.
 12. The composition of claim 11 wherein atleast about 10% of the MSCs comprise one or more nanoparticles, whereinthe nanoparticle comprises a gold or iron-oxide core, and wherein themajority of the one or more nanoparticles has a diameter in a range ofabout 0.1 nm to about 20 nm.
 13. The composition of claim 11 wherein thehigh-Z element is gold.
 14. The composition of claim 11 wherein at leastabout 70% of the cells are viable after a cryoprotective freeze-thawcycle.
 15. The composition of claim 14 wherein at least about 10% of theMSCs comprise one or more nanoparticles, wherein the nanoparticlecomprises a gold or iron-oxide core, and wherein the majority of the oneor more nanoparticles has a diameter in a range of about 0.1 nm to about20 nm.
 16. The composition of claim 14 wherein the nanoparticles are inan amount of at least 5 femtograms.
 17. The composition of claim 16 in atherapeutically effective amount and wherein the high-Z element is gold.18. The composition of claim 14 wherein the high-Z element is gold andwherein upon administration to a subject with a tumor or cancer followedby irradiation of the diseased tissue with therapeutic radiation, anincreased therapeutic efficacy is attained compared to irradiationalone.
 19. A method of treating a diseased tissue in a subjectcomprising a step of administering the composition of claim 11 to thesubject and a subsequent step of irradiating the diseased tissue withtherapeutic radiation, optionally wherein the diseased tissue releasesMSC chemo-attractants.
 20. A method of detecting a diseased tissue in asubject comprising a step of administering the composition of claim 11to the subject and a subsequent step of imaging the subject or a portionthereof and detecting the nanoparticles.